WO1998014568A1 - Human growth gene and short stature gene region - Google Patents

Abstract

Subject of the present invention is an isolated human nucleic acid molecule encoding polypeptides containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID NO:1 and having regulating activity on human growth. Three novel genes residing within the about 500 kb short stature critical region on the X and Y chromosome were identified. At least one of these genes is responsible for the short stature phenotype. The cDNA corresponding to this gene may be used in diagnostic tools, and to further characterize the molecular basis for the short stature-phenotype. In addition, the identification of the gene product of the gene provides new means and methods for the development of superior therapies for short stature.

Description

HUMAN GROWTH GENE AND SHORT STATURE GENE REGION

The present invention relates to the isolation, identification and characteπzation of newly identified human genes responsible for disorders relating to human growth, especiallv for short stature or Turner svndrome, as well as the diagnosis and therapy of such disorders

The isolated genomic DNA or fragments thereof can be used for pharmaceutical purposes or as diagnostic tools or reagents for identification or characteπzation of the genetic defect involved in such disorders Subject of the present invention are further human growth proteins (transcπption factors A. B and C) which are expressed after transcπption of said DNA into RNA or mRNA and which can be used in the therapeutic treatment of disorders related to mutations in said genes The invention further relates to appropπate cDNA sequences which can be used for the preparation of recombinant proteins suitable for the treatment of such disorders Subject of the invention are further plasmid vectors for the expression of the DNA of these genes and appropπate cells containing such DNAs It is a further subject of the present invention to provide means and methods for the genetic treatment of such disorders in the area of molecular medicine using an expression plasmid prepared by incorporating the DNA of this invention downstream from an expression promotor which effects expression in a mammalian host cell

Growth is one of the fundamental aspects in the development of an organism, regulated by a highly organised and complex system Height is a multifactoπal trait, influenced by both environmental and genetic factors Developmental malformations concermng body height are common phenomena among humans of all races With an incidence of 3 in 100, growth retardation resulting in short stature account for the large majority of inborn deficiencies seen in humans

With an incidence of 1 2500 life-born phenotypic females, Turner syndrome is a common chromosomal disorder (Rosenfeld et al , 1996) It has been estimated that 1-2% of all human conceptions are 45, X and that as many as 99 % of such fetuses do not come to term (Hall and Gilchπst, 1990, Robins, 1990) Significant clinical vaπability exists in the phenotype of persons with Turner syndrome (or Ullnch-Turner syndrome) (Ullrich, 1930. Turner. 1938) Short stature, however, is a consistent finding and together with gonadal dysgenesis considered as the lead svmptoms of this disorder Turner svndrome is a true multifactoπal disorder Both the embrvonic lethality, the short stature, gonadal dysgenesis and the characteπstic somatic features are thought to be due to monosomy of genes common to the X and Y chromosomes The diploid dosis of those X-Y homologous genes are suggested to be requested for normal human development Turner genes (or anti-Turner genes) are expected to be expressed in females from both the active and inactive X chromosomes or Y chromosome to ensure correct dosage of gene product Haploinsufficiency (deficiency due to only one active copy), consequently would be the suggested genetic mechanism underlying the disease

A vaπety of mechanisms underlying short stature have been elucidated so far Growth hormone and growth hormone receptor deficiencies as well as skeletal disorders have been described as causes for the short stature phenotype (Martial et al . 1979, Phillips et al , 1981, Leung et al , 1987, Goddard et al . 1995) Recently, mutations in three human fibroblast growth factor receptor-encoding genes (FGFR 1-3) were identified as the cause of vaπous skeletal disorders, including the most common form of dwarfism, achondroplasia (Shiang et al , 1994, Rousseau et al , 1994, Muenke and Schell, 1995) A well-known and frequent (1 2500 females) chromosomal disorder, Turner Syndrome (45,X), is also consistently associated with short stature Taken together, however, all these different known causes account for only a small fraction of all short patients, leaving the vast majoπty of short stature cases unexplained to date

The sex chromosomes X and Y are believed to harbor genes influencing height (Ogata and Matsuo, 1993) This could be deduced from genotype-phenotype correlations in patients with sex chromosome abnormalities Cytogenetic studies have provided evidence that terminal deletions of the short arms of either the X or the Y chromosome consistently lead to short stature m the respective individuals (Zuffardi et al , 1982, Curry et al , 1984) More than 20 chromosomal rearrangements associated with terminal deletions of chromosome Xp and Yp have been reported that localize the gene(s) responsible for short stature to the pseudoautosomal region (PAR1) (Ballabio et al , 1989, Schaefer et al , 1993) This localisation has been narrowed down to the most distal 700 kb of DNA of the PAR1 region, with DXYS15 as the flanking marker (Ogata et al , 1992. 1995)

Mammalian growth regulation is organized as a complex system It is conceivable that multiple growth promoting genes (proteins) interact with one another in a highlv organized way One of those genes controlling height has tentatively been mapped to the pseudoautosomal region PAR1 (Ballabio et al., 1989), a region known to be freely exchanged between the X and Y chromosomes (for a review see Rappold, 1993) The entire PAR1 region is approximately 2,700kb

The critical region for short stature has been defined with deletion patients. Short stature is the consequence when an entire 700kb region is deleted or when a specific gene within this critical region is present in haploid state, is interrupted or mutated (as is the case with idiotypic short stature or Turner sydrome). The frequency of Turner's syndrome is 1 in 2500 females worldwide; the frequency of this kind of idiopathic short stature can be estimated to be 1 in 4 000 - 5 000 persons Turner females and some short stature individuals usually receive an unspecific treatment with growth hormone (GH) for many years to over a decade although it is well known that they have normal GH levels and GH deficiency is not the problem The treatment of such patients is very expensive (estimated costs approximately 30 000 USD p. a ) Therefore, the problem existed to provide a method and means for distinguishing short stature patients on the one side who have a genetic defect in the respective gene and on the other side patients who do not have any genetic defect in this gene Patients with a genetic defect in the respective gene - either a complete gene deletion (as in Turner syndrome) or a point mutation (as in idiopathic short stature) - should be susceptible for an alternative treatment without human GH. which now can be devised

Genotype/phenotype correlations have supported the existence of a growth gene in the proximal part of Yq and in the distal part of Yp Short stature is also consistently found in individuals with terminal deletions of Xp Recently, an extensive search for male and female patients with partial monosomies of the pseudoautosomal region has been undertaken. On the basis of genotype-phenotype correlations, a minimal common region of deletion of 700 kb DNA adjacent to the telomere was determined (Ogata et al., 1992, Ogata et al , 1995) The region of interest was shown to lie between genetic markers DXYS20 (3cosPP) and DXYS15 (113D) and all candidate genes for growth control from within the PAR1 region (e.g , the hemopoietic growth factor receptor a, CSF2RA) (Gough et al., 1990) were excluded based on their physical location (Rappold et al., 1992) That is. the genes were within the 700 kb deletion region of the 2.700 kb PAR1 region Deletions of the pseudoautosomal region (PAR1 ) of the sex chromosomes were recently discovered in individuals with short stature and subsequently a minimal common deletion region of 700 kb within PAR1 was defined Southern blot analysis on DNA of patients AK and SS using different pseudoautosomal markers has identified an Xp terminal deletion of about 700 kb distal to DXYS15 (113D) (Ogata et al, 1992, Ogata et al, 1995)

The gene region coπesponding to short stature has been identified as a region of approximately 500 kb, preferably approximately 1 0 kb in the PAR1 region of the X and Y chromosomes Three genes in this region have been identified as candidates for the short stature gene These genes were designated SHOX (also referred to as SHOX93 or HOX93), (SHOX = short stature homeobox-containmg gene), pET92 and SHOT (SHOX-like homeobox gene on chromosome three) The gene SHOX which has two separate splicing sites resulting in two variations (SHOX a and b) is of particular importance In preliminary investigations, essential parts of the nucleotide sequence of the short stature gene could be analysed (SEQ ED No 8) Respective exons or parts thereof could be predicted and identified (e g exon I [G310], exon II [ET93], exon IV [G108], pET92) The obtained sequence information could then be used for designing appropriate primers or nucleotide probes which hybridize to parts of the SHOX gene or fragments thereof By conventional methods, the SHOX gene can then be isolated By further analysis of the DNA sequence of the genes responsible for short stature, the nucleotide sequence of exons I - V could be refined (v fig 1 - 3) The gene SHOX contains a homeobox sequence (SEQ ID NO 1 ) of approximately 180 bp (v fig 2 and fig 3), starting from the nucleotide coding for amino acid position 1 17 (Q) to the nucleotide coding for amino acid position 176 (E), i e from CAG (440) to GAG (619) The homeobox sequence is identified as the homeobox-pET93 (SHOX) sequence and two point mutations have been found in individuals with short stature in a German (Al) and a Japanese patient by screening up to date 250 individuals with idiopahtic short stature Both point mutations were found at the identical position and leading to a protein truncation at amino acid position 195, suggesting that there may exist a hot spot of mutation Due to the fact that both mutations found, which lead to a protein truncation, are at the identical position, it is possible that a putative hot spot of recombination exisits with exon 4 (G108) Exon specific primers can therefore be used as indicated below, e g GCA CAG CCA ACC ACC TAG (for) or TGG AAA GGC ATC ATC CGT AAG (rev) The above-mentioned novel homeobox-containing gene, SHOX, which is located within the 170 kb interval, is alternatively spliced generating two proteins with diverse function Mutation analysis and DNA sequencing were used to demonstrate that short stature can be caused by mutations in SHOX

The identification and cloning of the short stature critical region according to the present invention was performed as follows Extensive physical mapping studies on 15 individuals with partial monosomy in the pseudoautosomal region (PAR1) were performed By correlating the height of those individuals with their deletion breakpoints a short stature (SS) critical region of approximately 700 kb was defined This region was subsequently cloned as an overlapping cosmid contig using yeast artificial chromosomes (YACs) from PAR 1 (Ried et al.. 1996) and by cosmid walking To search for candidate genes for SS within this interval, a variety of techniques were applied to an approximately 600 kb region between the distal end of cosmid 56G10 and the proximal end of 5 ID 11 Using cDNA selection, exon trapping, and CpG island cloning, the two novel genes were identified

The position of the short stature critical interval could be refined to a smaller interval of 170 kb of DNA by characterizing three further specific individuals (GA, AT and RY), who were consistently short To precisely localize the rearrangement breakpoints of those individuals, fluorescence /// situ hybridization (FISH) on metaphase chromosomes was carried out using cosmids from the contig Patient GA. with a terminal deletion and normal height, defined the distal boundary of the critical region (with the breakpoint on cosmid 110E3), and patient AT, with an X chromosome inversion and normal height, the proximal boundary (with the breakpoint on cosmid 34F5) The Y-chromosomal breakpoint of patient RY, with a terminal deletion and short stature, was also found to be contained on cosmid 34F5, suggesting that this region contains sequences predisposing to chromosome rearrangements

The entire region, bounded by the Xp Yp telomere, has been cloned as a set of overlapping cosmids Fluorescence in situ hybridization (FISH) with cosmids from this region was used to study six patients with X chromosomal rearrangements, three with normal height and three with short stature Genotype-phenotype correlations narrowed down the critical short stature interval to 270 kb of DNA or even less as 170 kb, containing the gene or genes with an important role in human growth A minimal tiling path of six to eight cosmids bridging this interval is now available for interphase and metaphase FISH providing a valuable tool for diagnostic investigations on patients with idiopathic short stature

Brief Description of the Drawings

Figure 1 is a gene map of the SHOX gene including five exons which are identified as follows exon I G310, exon II. ET93, exon III ET45, exon IV Gl 08 and exons Va and Vb, whereby exons Va and Vb result from two different splicing sites of the SHOX gene Exon II and III contain the homeobox sequence of 180 nucleotides

Figures 2 and 3 are the nucleotide and predicted amino acid sequences of SHOXa and SHOXb

SHOX a: The predicted start of translation begins at nucleotide 92 with the first in-frame stop codon (TGA) at nucleotides 968 - 970, yielding an open reading frame of 876 bp that encodes a predicted protein of 292 amino acids (designated as transcription factor A or SHOXa protein, respectively) An in-frame, 5 stop codon at nucleotide 4, the start codon and the predicted termination stop codon are in bold The homeobox is boxed (starting from amino acid position 117 (Q) to 176 (E), i e CAG thru GAG in the nucleotide sequence) The locations of introns are indicated with arrows Two putative polyadenylation signals in the 3 untranslated region are underlined

SHOX b An open reading frame of 876 bp exists from A in the first methionin at nucleotide 92 to the in-frame stop codon at nucleotide 767-769, yielding an open reading frame of 675 bp that encodes a predicted protein of 225 amino acids (transcription factor B or SHOXb protein, respectively) The locations of introns are indicated with arrows Exons I-IV are identical with SHOXa, exon V is specific for SHOX b A putative polyadenylation signal in the 3' untranslated region is underlined

Figure 4 are the nucleotide and predicted amino acid sequence of SHOT The predicted start of translation begins at nucleotide 43 with the first in-frame stop codon (TGA) at nucleotides 613 - 615, yielding an open reading frame of 573 bp that encodes a predicted protein of 190 amino acids (designated as transcription factor C or SHOT protein, respectively) The homeobox is boxed (starting from amino acid position 1 1 (Q) to 70 (E), l e CAG thru GAG in the nucleotide sequence) The locations of introns are indicated with arrows Two putative polyadenylation signals in the 3 untranslated region are underlined

FigureS gives the exon/intron organization of the human SHOX gene and the respective positions in the nucleotide sequence

Brief Description of the SEQ ID

SEQ ED NO 1 translated amino acid sequence of the homeobox domain (180 bp) SEQ ID NO. 2 exon II (ET93) ofthe SHOX gene

SEQ ID NO 3 exon I (G310) of the SHOX gene

SEQ ID NO 4 exon III (ET45) of the SHOX gene

SEQ ID NO 5 exon IV (G108) of the SHOX gene

SEQ ID NO 6 exon Va of the SHOX gene SEQ ID NO 7 exon Vb of the SHOX gene

SEQ ID NO 8 preliminary nucleotide sequence of the SHOX gene

SEQ ID NO 9 ET92 gene

SEQ ID NO 10 SHOXa sequence (see also fig 2)

SEQ ID NO 1 1 transcription factor A (see also fig 2) SEQ ID NO 12 SHOXb sequence (see also fig 3)

SEQ ID NO 13 transcription factor B (see also fig 3)

SEQ ID NO 14 SHOX gene

SEQ ID NO 15 SHOT sequence (see also fig 4)

SEQ ID NO 16 transcription factor C (see also fig 4)

Since the target gene leading to disorders in human growth (e g short stature region) was unknown prior to the present invention, the biological and clinical association of patients with this deletion could give insights to the function of this gene In the present study, fluorescence in situ hybridization (FISH) was used to examine metaphase and inteφhase lymphocyte nuclei of six patients The aim was to test all cosmids of the overlapping set for their utility as FISH probes and to determine the breakpoint regions in all four cases, thereby determining the minimal critical region for the short stature gene

Duplication and deletion of genomic DNA can be technically assessed by carefully controlled quantitative PCR or dose estimation on Southern blots or by using RFLPs However, a particularly reliable method for the accurate distinction between single and double dose of markers is FISH, the clinical application of is presently routine Whereas in interphase FISH, the pure absence or presence of a molecular marker can be evaluated, FISH on metaphase chromosomes may provide a semi-quantitative measurement of inter-cosmid deletions The present inventor has determined that deletions of about 10 kb (25% of signal reduction) can still be detected This is of importance, as practically all disease genes on the human X chromosome have been associated with smaller and larger deletions in the range from a few kilobases to several megabases of DNA (Nelson et al , 1995)

Subject of the present invention are therefore DNA sequences or fragments thereof which are part of the genes responsible for human growth (or for short stature, respectively, in case of genetic defects in these genes) Three genes responsible for human growth were identified SHOX, pET92 and SHOT DNA sequences or fragments of these genes, as well as the respective full length DNA sequences of these genes can be transformed in an appropriate vector and transfected into cells When such vectors are introduced into cells in an appropriate way as they are present in healthy humans, it is devisable to treat diseases involved with short stature, i e Turners syndrome, by modern means of gene therapy For example, short stature can be treated by removing the respective mutated growth genes responsible for short stature It is also possible to stimulate the respective genes which compensate the action of the genes responsible for short stature, i e by inserting DNA sequences before, after or within the growth/short stature genes in order to increase the expression of the healthy allels By such modifications of the genes, the growth/short stature genes become activated or silent, respectively This can be accomplished by inserting DNA sequences at appropriate sites within or adjacent to the gene, so that these inserted DNA sequences interfere with the growth/short stature genes and thereby activate or prevent their transcription It is also devisable to insert a regulatory element (e g a promotor sequence) before said growth genes to stimulate the genes to become active It is further devisable to stimulate the respective promotor sequence in order to overexpress - in the case of Turner syndrome - the healthy functional allele and to compensate for the missing allele The modification of genes can be generally achieved by inserting exogenous DNA sequences into the growth gene / short stature gene via homologous recombination

The DNA sequences according to the present invention can also be used for transformation of said sequences into animals, such as mammals, via an appropriate vector svstem These transgemc animals can then be used for in vivo investigations for screening or identifying pharamceutical agents which are useful in the treatment of diseases involved with short stature If the ammals positively respond to the administration of a candidate compound or agent, such agent or compound or derivatives thereof would be devisable as pharmaceutical agents Bv appropπate means, the DNA sequences of the present invention can also be used in genetic experiments aiming at finding methods in order to compensate for the loss of genes responsible for short stature (knock-out ammals)

In a further object of this invention, the DNA sequences can also be used to be transformed into cells These cells can be used for identifying pharmaceutical agents useful for the treatment of diseases involved with short stature, or for screening of such compounds or library of compounds In an appropπate test svstem. vaπations in the phenotype or in the expression pattern of these cells can be determined, thereby allowing the identification of interesting candidate agents in the development of pharmaceutical drugs

The DNA sequences of the present invention can also be used for the design of appropπate pπmers which hvbπdize with segments of the short stature genes or fragments thereof under stringent conditions Appropπate pπmer sequences can be constructed which are useful in the diagnosis of people who have a genetic defect causing short stature In this respect it is noteworthv that the two mutations found occur at the identical position, suggesting that a mutational hot spot exists

In general, DNA sequences according to the present invention are understood to embrace also such DNA sequences which are degenerate to the specific sequences shown, based on the degeneracy of the genetic code, or which hvbπdize under stπngent conditions with the specifically shown DNA sequences

The present invention encompasses especially the following aspects

a) An isolated human nucleic acid molecule encoding polypeptides containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID NO 1 and having regulating activity on human growth b) An isolated DNA molecule comprising the nucleotide sequence essentially as indicated in fig. 2, fig 3 or fig 4, and especially as shown in SEQ ED NO 10, SEQ ID NO 12 or SEQ ID NO 15 c) DNA molecules capable of hybridizing to the DNA molecules of item b) d) DNA molecules of item c) above which are capable of hybridization with the DNA molecules of item 2 under a temperature of 60 - 70 °C and in the presence of a standard buffer solution e) DNA molecules comprising a nucleotide sequence having a homology of seventy percent or higher with the nucleotide sequence of SEQ ID NO' 10, SEQ ED NO 12 or SEQ ID NO 15 and encoding a polypeptide having regulating activity on human growth f) Human growth proteins having the amino acid sequence of SEQ ED NO 1 1, 13 or 16 or a functional fragment thereof g) Antibodies obtained from immunization of ammals with human growth proteins of item f) or antigenic variants thereof h) Pharmaceutical compositions comprising human growth proteins or functional fragments thereof for treating disorders caused by genetic mutations of the human growth gene i) A method of screening for a substance effective for the treatment of disorders mentioned above under item h) comprising detecting messenger RNA hybridizing to any of the DNA molecules decribed in a) - e) so as to measure any enhancement in the expression levels of the DNA molecule in response to treatment of the host cell with that substance j) An expression vector or plasmid containing any of the nucleic acid molecules described in a) - e) above which enables the DNA molecules to be expressed in mammalian cells k) A method for the determination of the gene or genes responsible for short stature in a biological sample of body tissues or body fluids

In the method k) above, preferably nucleotide amplification techniques, e.g PCR, are used for detecting specific nucleotide sequences known to persons skilled in the art, and described, for example, by Mullis et al 1986, Cold Spring Harbor Symposium Quant Biol 51, 263-273, and Saiki et al , 1988, Science 239, 487-491, which are incorporated herein by reference The short stature nucleotide sequences to be determined are mainly those represented by sequences SEQ ED No 2 to SEQ ED No 7 In principle, all oligonucleotide primers and probes for amplifying and detecting a genetic defect responsible for deminished human growth in a biological sample are suitable for amplifying a target short stature associated sequence. Especially, suitable exon specific primer pairs according to the invention are provided by table 1. Subsequently, a suitable detection, e.g. a radioactive or non-radioactive label is carried out.

Table 1 :

Exon Sense primer Antisense primer Product (bp) Ta (°C)

5'-I (G310) SP 1 ASP 1 194 58

3'-I (G310) SP 2 ASP 2 295 58

II (ET93) SP 3 ASP 3 262 76/72/68

III (ET45) SP 4 ASP 4 120 65

IV (G108) SP 5 ASP 5 154 62

Va (SHOXa) SP 6 ASP 6 265 61

explanation of the abbreviations for the primers:

SP 1 ATTTCCAATGGAAAGGCGTAAATAAC SP2 ACGGCTTTTGTATCCAAGTCTTTTG SP3 GCCCTGTGCCCTCCGCTCCC SP4 GGCTCTTCACATCTCTCTCTGCTTC SP5 CCACACTGACACCTGCTCCCTTTG SP6 CCCGCAGGTCCAGGCTCAGCTG ASP1 CGCCTCCGCCGTTACCGTCCTTG

ASP 2 : CCCTGGAGCCGGCGCGCAAAG ASP 3 CCCCGCCCCCGCCCCCGG ASP 4 CTTCAGGTCCCCCCAGTCCCG

ASP 5 : CTAGGGATCTTCAGAGGAAGAAAAAG ASP 6 GCTGCGCGGCGGGTCAGAGCCCCAG Also, a single stranded RNA can be used as target Methods for reversed transcribing RNA into cDNA are also well known and described in Sambrook et al , Molecular Cloning A Laboratory Manual, New York, Cold Spring Harbor Laboratory 1989 Alternatively, preferred methods for reversed transcription utilize thermostable DNA polymerases having RT activity

Further, the technique described before can be used for selecting those person from a group of persons being of short stature characterized by a genetic defect and which allows as a consequence a more specific medical treatment

In another subject of the present invention, the transcription factors A. B and C can be used as pharmaceutical agents These transcription factors initiate a still unknown cascade of biological effects on a molecular level involved with human growth These proteins or functional fragments thereof have a mitogenic effect on various cells Especially, they have an osteogenic effect They can be used in the treatment of bone diseases, such as e g osteoporosis, and especially all those diseases involved with disturbance in the bone calcium regulation

As used herein, the term „isolated" refers to the original derivation of the DNA molecule by cloning It is to be understood however, that this term is not intended to be so limiting and. in fact, the present invention relates to both naturally occurring and synthetically prepared seqences. as will be understood by the skilled person in the art

The DNA molecules of this invention may be used in forms of gene therapy involving the use of an expression plasmid prepared by incoφorating an appropriate DNA sequence of this invention downstream from an expression promotor that effects expression in a mammalian host cell Suitable host cells are procaryotic or eucaryotic cells Procaryotic host cells are, for example, E coli, Bacillus subtilis, and the like. By transfecting host cells with replicons originating from species adaptable to the host, that is, plasmid vectors containing replication starting point and regulator sequences, these host cells can be transfected with the desired gene or cDNA Such vectors are preferably those having a sequence that provides the transfected cells with a property (phenotype) by which they can be selected For example, for E coli hosts the strain E coli K12 is typically used, and for the vector either pBR322 or pUC plasmids can be generally employed Examples for suitable promotors for E coli hosts are tφ promotor. lac promotor or lpp promotor

If desired, secretion of the expression product through the cell membrane can be effected by connecting a DNA sequence coding for a signal peptide sequence at the 5 ^' upstream side of the gene. Eucaryotic host cells include cells derived from vertebrates or yeast etc As a vertebrate host cell, COS cells can be used (Cell, 1981, 23 175 - 182), or CHO cells Preferably, promotors can be used which are positioned 5 ^' upstream of the gene to be expressed and having RNA splicing positions, polyadenylation and transcription termination seqences

The transcription factors A, B and C of the present invention can be used to treat disorders caused by mutations in the human growth genes and can be used as growth promoting agents Due to the polymorphism known in the case of eukaryotic genes, one or more amino acids may be substituted Also, one or more amino acids in the polypeptides can be deleted or inserted at one or more sites in the amino acid sequence of the polypeptides of SEQ ED NO 1 1, 13 or 16 Such polypeptides are generally referred to equivalent polypeptides as long as the underlying biological acitivity of the unmodified polypeptide remains essentially unchanged

The present invention is illustrated by the following examples

Example 1 Patients

All six patients studied had de novo sex chromosome aberrations

CC is a girl with a karyotype 45.X/46,X psu die (X) (Xqter → Xp22 3 Xp22 3 → Xqter) At the last examination at 6 1/2 years of age, her height was 1 14 cm (25 - 50 the % percentile) Her mother's height was 155 cm, the father was not available for analysis For details, see Henke et al., 1991

GA is a girl with a karyotype 46,X der X (3pter → 3p23 Xp22.3 — > Xqter) At the last examination at 17 years, normal stature (159 cm) was observed Her mother's height is 160 cm and her father's height 182 cm For details, see Kulharya et al, 1995

SS is a girl with a karyotype 46,X rea (X) (Xqter → Xq26 Xp22 3 → Xq26 ) At 11 years her height remained below the 3rd percentile growth curve for Japanese girls, her predicted adult height (148 5 cm) was below her target height (163 cm) and target range (155 to 191 em) For details, see Ogata et alt, 1992 AK is a girl with a karyotype 46,X rea (X) (Xqter → Xp22 3 Xp22.3 → Xp21.3 ) At 13 years her height remained below the 2nd percentile growth curve for Japanese girls, her predicted adult height (142 8 cm) was below her target height (155 5 cm) and target range (147 5 - 163.5 em) For details, see Ogata et alt, 1995

RY the karyotype of the ring Y patient is 46,X,r(Y)/46,Xdic r(Y)/45,X[95 3.2], as examined on 100 lymphocytes; at 16 years of age his final height was 148; the heights of his three brothers are all in the normal range with 170 cm (16 years, brother 1 ), 164 cm ( 14 years, brother 2) and 128 cm (9 years, brother 3), respectively Growth retardation of this patient is so severe that it would also be compatible with an additional deletion of the GCY locus on Yq

AT boy with ataxia and inv(X). normal height of 116 cm at age 7, parents^' heights are 156 cm and 190 cm. respectively

Patients for mutation analysis

250 individuals with idiopathic short stature were tested for mutations in SHOXa The patients were selected on the following criteria height for chronological age was below the 3rd centile of national height standards, minus 2 standard deviations (SDS). no causative disease was known, in particular- normal weight (length) for gestational age, normal body proportions, no chronic organic disorder, normal food intake, no psychiatric disorder, no skeletal dysplasia disorder, no thyroid or growth hormone deficiency

Family A Cases 1 and 2 are short statured children of a German non-consanguineous family The boy (case 1) was born at the 38th week of gestation by cesarian section Birth weight was 2660 g, birth length 47 cm. He developed normally except for subnormal growth On examination at the age of 6 4 years, he was proportionate small (106.8 cm, -2 6 SDS) and obese (22 7 kg), but otherwise normal His bone age was not retarded (6 yrs) and bone dysplasia was excluded by X-ray analysis IGF-I and IGFBP-3 levels as well as thyroid parameters in serum rendered GH or thyroid hormone deficiency unlikely The girl (case 2) was born at term by cesarian section. Birth weight was 2920 g, birth length 47 cm Her developmental milestones were normal, but by the age of 12 months poor growth was apparent (length 67 cm, -3 0 SDS) At 4 years she was 89 6 cm of height (- 3 6 SDS) No dysmorphic features or dysproportions were apparent. She was not obese (13 kg). Her bone age was 3 5 years and bone dysplasia was excluded Hormone parameters were normal It is interesting to note that both the girl and the boy grow on the 50 percentile growth curve for females with Turner syndrome. The mother is the smallest of the family and has a mild rhizomelic dysproportion (142.3 cm, -3 8 SDS) One of her two sisters (150 cm, -2 5 SDS) and the maternal grandmother (153 cm, -2 0 SDS) are all short without any dysproportion One sister has normal stature (167 cm, +0 4 SDS) The father's height is 166 cm (-1 8 SDS) and the maternal grandfather' height is 165 cm (-1 9 SDS) The other patient was of Japanese origin and showed the identical mutation

Example 2 Identification of the short stature gene

A In situ hybridization

a) Florescence in situ hybridization (FISH)

Florescence in situ hybridization (FISH) using cosmids residing in the Xp/Yp pseudoautosomal region (PAR1 ) was carried out FISH studies using cosmids 64/75cos (LLNLcl 10H032), E22cos (2e2). Fl/14cos (1 10A7), Ml/70cos (110E3), P99F2cos (43C11 ), P99cos (LLNLcl 10P2410). Bόcosb (lCRFcl04H0425), F20cos (34F5), F21cos (ICRFcl04G041 1 ), F3cos2 (9E3), F3cosl ( 1 1E6), P117cos (29B1 1), Pόcosl (ICRFcl04P01 17), P6cos2 (LLNLcl 10E0625) and E4cos (15G7) was carried out according to published methods (Lichter and Cremer. 1992) In short, one microgram of the respective cosmid clone was labeled with biotin and hybridized to human metaphase chromosomes under conditions that suppress signals from repetitive DNA sequences Detection of the hybridization signal was via FITC-conjugated avidin Images of FITC were taken by using a cooled charge coupled device camera system (Photometries, Tucson, AZ)

b) Physical mapping

Cosmids were derived from Lawrence Livermore National Laboratory X- and Y- chromosome libraries and the Imperial Cancer Research Fund London (now Max Planck Institute for Molecular Genetics Berlin) X chromosome library Using cosmids distal to DXYS15. namely E4cos, P6cos2. Pόcosl, P117cos and F3cosl one can determine that two copies are still present of E4cos, P6cos2, Pόcosl and one copy of P117cos and F3cosl Breakpoints of both patients AK and SS map on cosmid Pόcosl, with a maximum physical distance of 10 kb from each other. It was concluded that the abnormal X chromosomes of AK and SS have deleted about 630 kb of DNA.

Further cosmids were derived from the ICRF X chromosome specific cosmid library (ICRFcl04), the Lawrence Livermore X chromosome specific cosmid library (LLNLcl 10) and the Y chromosome specific library (LLC03'M'), as well as from a self- made cosmid library covering the entire genome. Cosmids were identified by hybridisation with all known probes mapping to this region and by using entire YACs as probes. To verify overlaps, end probes from several cosmids were used in cases in which overlaps could not be proven using known probes.

c) Southern Blot Hybridisation

Southern blot analysis using different pseudoautosomal markers has provided evidence that the breakpoint on the X chromosome of patient CC resides between DXYS20 (3cosPP) and DXYS60 (U7A) (Henke et al, 1991). In order to confirm this finding and to refine the breakpoint location, cosmids 64/75cos, E22cos, Fl/14cos, Ml/70cos. F2cos, P99F2cos and P99cos were used as FISH probes. The breakpoint location on the abnormal X of patient CC between cosmids 64/75cos (one copy) and Fl/14cos (two copies) on the E22PAC could be determined Patient CC with normal stature consequently has lost approximately 260-290 kb of DNA.

Southern blot hybridisations were carried out at high stringency conditions in Church buffer (0.5 M NaPi pH 7.2, 7% SDS. ImM EDTA) at 65°C and washed in 40 mM NaPi, 1% SDS at 65°C.

d) FISH Analysis

Biotinylated cosmid DNA (insert size 32 - 45 kb) or cosmid fragments (10 - 16 kb) were hybridised to metaphase chromosomes from stimulated lymphocytes of patients under conditions as described previously (Lichter and Cremer, 1992). The hybridised probe was detected via avidin-conjugated FITC.

e) PCR Amplification

All PCRs were performed in 50 μl volumes containing 100 pg-200 ng template, 20 pmol of each primer, 200 μM dNTP's (Pharmacia), 1.5 mM MgCl₂, 75 mMTris/HCl pH9, 20mM (NHO₂SO4 0.01% (w/v) Tween20 and 2 U of Goldstar DNA Polymerase (Eurogentec). Thermal cycling was carried out in a Thermocycler GeneE (Techne). f Exon Amplification

Four cosmid pools consisting of each four to five clones from the cosmid contigs were used for exon amplification expeπments The cosmids in each cosmid pool were partially digested with Sau3A Gel purified fractions in the size range of 4-10 kb were cloned in the BamHI digested pSPL3B vector (Burn et al, 1995) and used for the exon amplification expeπments as previously described (Church et al , 1994)

g) Genomic Sequencing

Somficated fragments of the two cosmids LLOYNCO3'M' 15D10 and LLOYNC03'M'34F5 were subcloned separately into M13mpl8 vectors From each cosmid library at least 1000 plaques were picked. Ml 3 DNA prepared and sequenced using dve-terminators. Thermo Sequenase (Amersham) and universal M13-pπmer (MWG-BioTech) The gels were run on ABI-377 sequencers and data were assembled and edited with the GAP4 program (Staden)

Of all six patients, GA had the least well characterized chromosomal breakpoint The most distal markers previously tested for their presence or absence on the X were DXS1060 and DXS996, which map approximately 6 Mb from the telomere (Nelson et al . 1995) Several cosmids containing different gene sequences from within PAR1 (MIC2, ANT3, CSF2RA, and XE7) were tested and all were present on the translocation chromosome Cosmids from within the short stature critical region e g , chromosome, thereby placing the translocation breakpoint on cosmid Ml/70cos A quantitative compaπson of the signal intensities of Ml/70cos between the normal and the rearranged X indicates that approximately 70% of this cosmid is deleted

TABLE 2

CC GA AK ss

64/75cos - -

E22cos - -

Fl/14cos + -

Ml/70cos + (+)

F2cos +

P99F2cos + -

P99cos + -

Bόcos -»-

F20cos

F21cos

F3cos2

F3cosl - -

P1 17cos - -

Pόcosl +

P6cos2 4-

E4cos →- -

Table 2: This table summarizes the FISH data for the 16 cosmids tested on four patients [ - ] one copy, indicates that the respective cosmid was deleted on the rearranged X, but present on the normal X chromosome [ + ] two copies, indicates that the respective cosmid is present on the rearranged and on the normal X chromosome [(+)] breakpoint region, indicates that the breakpoint occurs within the cosmid as shown bv FISH

In summary, the molecular analysis on six patients with X chromosomal rearrangements using florescence-labeled cosmid probes and in situ hybridization indicates that the short stature critical region can be narrowed down to a 270 kb interval, bounded by the breakpoint of patient GA from its centromere distal side and by patients AK and SS on its centromere proximal side

Genotype-phenotype correlations may be informative and have been chosen to delineate the short stature cπtical interval on the human X and Y chromosome In the present study FISH analysis was used to study metaphase spreads and interphase nuclei of lymphocytes from patients carrying deletions and translocations on the X chromosome and breakpoints within Xp22 3 These breakpoints appear to be clustered in two of the four patients (AK and SS) presumably due to the presence of sequences predisposing to chromosome rearrangements One additional patient Ring Y has been found with an interruption in the 270 kb critical region, thereby reducing the cπtical interval to a 170 kb region

By correlating the height of all six individuals with their deletion breakpoint, an interval of 170 kb was mapped to within the pseudoautosomal region, presence or absence of which has a significant effect on stature This interval is bounded by the X chromosomal breakpoint of patient GA at 340 kb from the telomere (Xptel) distally and by the breakpoints of patients AT and RY at 510/520 kb Xptel proximally This assignment constitutes a considerable reduction of the cntical interval to almost one fourth of its previous size (Ogata et al , 1992, Ogata et al , 1995) A small set of six to eight cosmids are now available for FISH expeπments to test for the prevalence and significance of this genomic locus on a large series of patients with idiopathic short stature

B Identification of the Candidate Short Stature Gene

To search for transcπption units within the smallest 170 kb critical region, exon trapping and cDNA selection on six cosmids (110E3, F2cos. 43C1 1, P2410, 15D10, 34F5) was carried out Three different positive clones (ET93, ET45 and G108) were isolated by exon trapping, all of which mapped back to cosmid 34F5 Previous studies using cDNA selection protocols and an excess of 25 different cDNA libraπes had proven unsuccessful, suggesting that genes in this interval are expressed at very low abundancy

To find out whether any gene in this interval was missed, the nucleotide sequence of about 140 kb from this region of the PAR1 was determined, using the random M13 method and dye terminator chemistry The cosmids for sequence analysis were chosen to minimally overlap with each other and to collectively span the cπtical interval DNA sequence analysis and subsequent protein prediction by the "X Grail" program, version 1 3c as well as by the exon-trapping program FEXHB were carried out and confirmed all 3 previously cloned exons No protein-coding genes other than the previously isolated one could be detected

C Isolation of the Short Stature Candidate Gene SHOX

Assuming that all three exon clones ET93, ET45 and G108 are part of the same gene, they were used collectively as probes to screen 14 different cDNA libraries from 12 different fetal (lung, liver, brain 1 and 2) and adult tissues (ovary, placenta 1 and 2, fibroblast, skeletal muscle, bone marrow, brain, brain stem, hypothalamus, pituitary) Not a single clone among approximately 14 million plated clones was detected To isolate the full-length transcript. 3 ^' and 5'RACE were carried out For 3 'RACE, primers from exon G108 were used on RNA from placenta, skeletal muscle and bone marrow fibroblasts, tissues where G108 was shown to be expressed in Two different 3'RACE clones of 1173 and 652 bp were derived from all three tissues, suggesting that two different 3 'exons a and b exist The two different forms were termed SHOXa and SHOXb

To increase chances to isolate the complete 5'portion of a gene known to be expressed at low abundancy, a Hela cell line was treated with retinoic acid and phorbol ester PMA RNA from such an induced cell line and RNA from placenta and skeletal muscle were used for the construction of a 'Marathon cDNA library' Identical 5 'RACE cDNA clones were isolated from all three tissues

Experimental procedure

RT-PCR and cDNA Library Construction

Human polyAHNA of heart, pancreas, placenta, skeletal muscle, fetal kidney and liver was purchased from Clontech Total RNA was isolated from a bone marrow fibroblast cell line with TRIZOL reagent (Gibco-BRL) as described by the manufacturer First strand cDNA synthesis was performed with the Superscript first strand cDNA synthesis kit (Gibco-BRL) starting with 100 ng polyAHNA or lOμg total RNA using oligo(dt)- adapter primer (GGCCACGCGTCGACTAGTAC[dT]₂₀N After first strand cDNA synthesis the reaction mix was diluted 1/10. For further PCR experiments 5μl of this dilutions were used A 'Marathon cDNA library' was constructed from skeletal muscle and placenta polyA^'RNA with the marathon cDNA amplification kit (Clontech) as described by the manufacturer.

Fetal brain (catalog # HL5015b), fetal lung (JEL3022a), ovary (HL1098a), pituitary gland (FEL1097v) and hypothalamus (HL 1172b) cDNA libraries were purchased from Clontech. Brain, kidney, liver and lung cDNA libraries were part of the quick screen human cDNA library panel (Clontech). Fetal muscle cDNA library was obtained from the UK Human Genome Mapping Project Resource Center.

D. Sequence Analysis and Structure of SHOX Gene

A consensus sequence of SHOXa and SHOXb ( 1349 and 1870 bp) was assembled by analysis of sequences from the 5' and 3 'RACE derived clones. A single open reading frame of 1870 bp (SHOXa) and 1349 bp (SHOXb) was identified, resulting in two proteins of 292 (SHOXa) and 225 amino acids (SHOXb). Both transcripts a and b share a common 5'end, but have a different last 3 'exon, a finding suggestive of the use of alternative splicing signals. A complete alignment between the two cDNAs and the sequenced genomic DNA from cosmids LL0YNCO3"M"15D10 and LL0YNC3"M"34F5 was achieved, allowing establishment of the exon-intron structure (Fig.4) The gene is composed of 6 exons ranging in size from 58 bp (exon III) to 1146 bp (exon Va). Exon I contains a CpG-island, the start codon and the 5^" region. A stop codon as well as the 3^"-noncoding region is located in each of the alternatively spliced exons Va and Vb.

Example 3

Two cDNAs have been identified which map to the 160 kb region identified as critical for short stature. These cDNAs correspond to the genes SHOX and pET92. The cDNAs were identified by the hybridization of subclones of the cosmids to cDNA libraries.

Employing the set of cosmid clones with complete coverage of the critical region has now provided the genetic material to identify the causative gene. Positional cloning projects aimed at the isolation of the genes from this region are done by exon trapping and cDNA selection techniques By virtue of their location within the pseudoautosomal region, these genes can be assumed to escape X-inactivation and to exert a dosage effect

The clomng of the gene leading to short stature when absent (haploid) or deficient, represents a further step forward in diagnostic accuracy, providing the basis for mutational analysis withm the gene by e g single strand conformation polymorphism (SSCP) In addition, clomng of this gene and its subsequent biochemical characteπzation has opened the way to a deeper understanding of biological processes involved in growth control

The DNA sequences of the present invention provide a first molecular test to identify individuals with a specific genetic disorder within the complex heterogeneous group of patients with idiopathic short stature

Example 4 Expression Pattern of SHOXa and SHOXb

Northern blot analysis using single exons as hvbπdisation probes revelled a different expression profile for every exon, strongly suggesting that the bands of different size and intensities represent cross-hybπdisation products to other G,C πch gene sequences To achieve a more realistic expression profile of both genes SHOXa and b, RT-PCR expeπments on RNA from different tissues were carried out Whereas expression of SHOXa was observed in skeletal muscle, placenta, pancreas, heart and bone marrow fibroblasts, expression of SHOXb was restπcted to fetal kidney skeletal muscle and bone marrow fibroblasts, with the far highest expression in bone marrow fibroblasts

The expression of SHOXa in several cDNA libraπes made of fetal brain, lung and muscle, of adult brain, lung and pituitary and of SHOXb in none of the tested libraπes gives additional evidence that one spliced form (SHOXa) is more broadly expressed and the other (SHOXb) expressed in a predominantly tissue-specific manner

To assess the transcπptional activity of SHOXa and SHOXb on the X and Y chromosome we used RT-PCR of RNA extracted from various cell lines containing the active X, the inactive X or the Y chromosome as the onlv human chromosomes All cell lines revealed an amplification product of the expected length of 1 19 bp (SHOXa) and 541 bp (SHOXb), providing clear evidence that both SHOXa and b escape X- inactivation

SHOXa and SHOXb encode novel homeodomain proteins SHOX is highly conserved across species from mammalian to fish and flies The very 5' end and the very 3' end - besides the homeodomain- are likely conserved regions between man and mouse, indicating a functional significance Differences in those amino acid regions have not been allowed to accumulate during evolution between man and mouse

Experimental procedures

a) 5^" and 3 'RACE

To clone the 5^" end of the SHOXa and b transcripts, 5 'RACE was performed using the constructed 'Marathon cDNA libraries' The following oligonucleotide primers were used SHOX B rev, GAAAGGCATCCGTAAGGCTCCC (position 697-718, reverse strand [r]) and the adaptor primer API PCR was carried out using touchdown parameters 94°C for 2 min, 94°C for 30 sec. 70°C for 30 sec, 72°C for 2 min for 5 cycles 94°C for 30 sec, 66°C for 30 sec, 72°C for 2 min for 5 cycles 94°C for 30 sec, 62°C for 30 sec, 72°C for 2 min for 25 cycles A second round of amplification was performed usmg 1/100 of the PCR product and the following nested oligonucleotide primers SHOX A rev, GACGCCTTTATGCATCTGATTCTC (position 617-640 r) and the adaptor primer AP2 PCR was carried out for 35 cycles with an annealing temperature of 60°C

To clone the 3' end of the SHOXa and b transcripts, 3 'RACE was performed as previously described (Frohman et al , 1988) using oligo(dT)adaptor primed first strand cDNA The following oligonucleotide primers were used SHOX A for, GAATCAGATGCATAAAGGCGTC (position 619-640) and the oligo(dT)adaptor PCR was carried out using following parameters 94°C for 2 min, 94°C for 30 sec, 62°C for 30 sec, 72°C for 2 min for 35 cycles A second round of amplification was performed using 1/100 of the PCR product and the following nested oligonucleotide primers SHOX B for, GGGAGCCTTACGGATGCCTTTC (position 697-718) and the ohgo(dT)adaptor PCR was carried out for 35 cycles with annealing temperature of 62°C To validate the sequences of SHOXa and SHOXb transcripts, PCR was performed with a 5' oligonucleotide primer and a 3' oligonucleotide primer For SHOXa the following primers were used G310 for, AGCCCCGGCTGCTCGCCAGC (position 59-78) and SHOX D rev, CTGCGCGGCGGGTCAGAGCCCCAG (position 959-982 r) For SHOXb the following primers were used G310 for, AGCCCCGGCTGCTCGCCAGC and SHOX2A rev, GCCTCAGCAGCAAAGCAAGATCCC (position 1215-1238 r) Both PCRs were carried out using touchdown parameters 94°C for 2 min. 94°C for 30 sec, 70°C for 30 sec, 72°C for 2 min for 5 cycles 94°C for 30 sec. 68°C for 30 sec, 72°C for 2 min for 5 cycles 94°C for 30 sec, 65°C for 30 sec, 72°C for 2 min for 35 cycles Products were gel-purified and cloned for sequencing analysis

b) SSCP Analysis

SSCP analysis was performed on genomic amplified DNA from patients according to a previously described method (Orita et al , 1989) One to five μl of the PCR products were mixed with 5 μl of denaturation solution containing 95% Formamid and lOmM EDTA pH8 and denaturated at 95°C for 10 min Samples were immediately chilled on ice and loaded on a 10% Polyacryamidgel (Acrylamide Bisacryamide = 37 5 1 and 29 1, Multislotgel, TGGE base, Qiagen) containing 2% glycerol and lxTBE Gels were run at 15°C with 500V for 3 to 5 hours and silver stained as described in TGGE handbook (Qiagen. 1993)

c) Cloning and Sequencing of PCR Products

PCR products were cloned into pMOSBlue using the pMOSBlueT- Vector Kit from Amersham Overnight cultures of single colonies were lysed in 100 μl H₂0 by boiling for 10 min The lysates were used as templates for PCRs with specific primers for the cloned PCR product SSCP of PCR products allowed the identification of clones containing different alleles The clones were sequenced with CY5 labelled vector primers Uni and T7 by the cycle sequencing method described by the manufacturer (ThermoSequenase Kit (Amersham)) on an ALF express automated sequencer (Pharmacia)

d) PCR Screening of cDNA Libraries

To detect expression of SHOXa and b, a PCR screening of several cDNA libraries and first strand cDNAs was carried out with SHOXa and b specific primers For the cDNA libraries a DNA equivalent of 5x10⁸ pfu was used For SHOXa, primers SHOX E rev, GCTGAGCCTGGACCTGTTGGAAAGG (position 713-737 r) and SHOX a for were used For SHOXb. the following primers were used SHOX B for and SHOX2A rev Both PCRs were carried out using touchdown parameters 94°C for 2 min; 94°C for 30 sec, 68°C for 30 sec, 72°C for 40 sec for 5 cycles. 94°C for 30 sec, 65°C for 30 sec, 72°C for 40 sec for 5 cycles 94°C for 30 sec, 62°C for 30 sec, 72°C for 40 sec for 35 cycles.

e) PCR Screening of cDNA Libraries

To detect expression of SHOXa and b, a PCR screening of several cDNA libraries and first strand cDNAs was carried out with SHOXa and b specific primers For the cDNA libraries a DNA equivalent of 5x10⁸ pfu was used For SHOXa, primers SHOX E rev. GCTGAGCCTGGACCTGTTGGAAAGG (position 713-737 r) and SHOX a for were used. For SHOXb. the following primers were used SHOX B for and SHOX2A rev Both PCRs were carried out using touchdown parameters 94°C for 2 min. 94°C for 30 sec. 68°C for 30 sec. 72°C for 40 sec for 5 cycles 94°C for 30 sec, 65°C for 30 sec. 72°C for 40 sec for 5 cycles 94°C for 30 sec. 62°C for 30 sec. 72°C for 40 sec for 35 cycles

Example 5

Expression pattern of OG12, the putative mouse homolog of both SHOX and SHOT

In situ hybridisation on mouse embryos ranging from day 5 p c and day 18,5 p c , as well as on fetal and newborn animals was carried out to establish the expression pattern Expression was seen in the developing limb buds, in the mesoderm of nasal processes which contribute to the formation of the nose and palate, in the eyelid, in the aorta, in the developing female gonads, in the developing spinal cord (restricted to differentiating motor neurons) and brain. Based on this expression pattern and on the mapping position of its human homolog SHOT, SHOT represents a likely candidate for the Cornelia de Lange syndrome which includes short stature

Example 6

Isolation of a novel SHOX-like homeobox gene on chromosome three, SHOT, being related to human growth / short stature

A new gene called SHOT (for SHOX-homolog on chromosome three) was isolated in human, sharing the most homology with the murine OG12 gene and the human SHOX gene The human SHOT gene and the murine OG12 genes are highly homologous, with 99 % identity at the protein level Although not yet proven, due to the striking homology between SHOT and SHOX ( identity within the homeodomain only), it is likely that SHOT is also a gene likely involved in short stature or human growth

SHOT was isolated using primers from two new human ESTs (HS 1224703 and HS 126759) from the EMBL database, to amplify a reverse-transcribed RNA from a bone marrow fibroblast line (Rao et al, 1997) The 5 and 3 ^' ends of SHOT were generated by RACE-PCR from a bone marrow fibroblast library that was constructed according to Rao et al , 1997 SHOT was mapped by FISH analysis to chromosome 3q25/q26 and the murine homolog to the syntenic region on mouse chromosome 3 Based on the expression pattern of OG12. its mouse homolog, SHOT represents a candidate for the Cornelia Lange syndrome (which shows short stature and other features, including craniofacial abnormalities) mapped to this chromosomal interval on 3q25/26

Example 7

Searching for Mutations in Patients with Idiopathic Short Stature

The DNA sequences of the present invention are used in PCR, LCR. and other known technologies to determine if such individuals with short stature have small deletions or point mutations in the short stature gene

A total of initially 91 (in total 250 individuals) unrelated male and female patients with idiopathic short stature (idiopathic short stature has an estimated incidence of 2 - 2.5 % in the general population) were tested for small rearrangements or point mutations in the SHOXa gene Six sets of PCR primers were designed not only to amplify single exons but also sequences flanking the exon and a small part of the 5'UTR For the largest exon, exon one, two additional internal-exon primers were generated Primers used for PCR are shown in table 2

Single strand conformation polymoφhism (SSCP) of all amplified exons ranging from 120 to 295 bp in size was carried out Band mobility shifts were identified in only 2 individuals with short stature (Y91 and Al) Fragments that gave altered SSCP patterns (unique SSCP conformers) were cloned and sequenced To avoid PCR and sequencing artifacts, sequencing was performed on two strands using two independent PCR reactions The mutation in patient Y91 resides 28bp 5 'of the start codon in the 5'UTR and involves a cytidme-to-guanine substitution To find out if this mutation represents a rare polymorphism or is responsible for the phenotype by regulating gene expression e g though a weaker binding of translation initiation factors, his parents and a sister were tested As both the sister and father with normal height also show the same SSCP variant (data not shown), this base substitution represents a rare polymorphism unrelated to the phenotype

Cloning and sequencing of a unique SSCP conformer for patient Al revealed a cytidine- to-thvmidine base transition (nucleotide 674) which introduces a termination codon at amino-acid position 195 of the predicted 225 and 292 ammo-acid sequences, respectively To determine whether this nonsense mutation is genetically associated with the short stature in the family, pedigree analysis was carried out It was found that all six short individuals (defined as height belov, 2 standard deviations) showed an aberrant SSCP shift and the cvtidine-to-thymidine transition Neither the father, nor one aunt and maternal grandfather with normal height showed this mutation, indicating that the grandmother has transferred the mutated allele onto two of her daughters and her two grandchildren Thus, there is concordance between the presence of the mutant allele and the short stature phenotype in this family

The identical situation as indicated above was found in another short stature patient of Japanese origin

Example 8

The DNA sequences of the present invention are used to characterize the function of the gene or genes The DNA sequences can be used as search queries for data base searching of nucleic acid or amino acid databases to identify related genes or gene products The partial amino acid sequence of SHOX93 has been used as a search query of amino acid databases The search showed very high homology to manv known homeobox proteins The cDNA sequences of the present invention can be used to recombmantly produce the peptide Vaπous expression systems known to those skilled in the art can be used for recombinant protein production

By conventional peptide synthesis (protein synthesis according to the Merπfield method), a peptide having the sequence CSKSFDQKSKDGNGG was synthesized and polyclonal antibodies were derived in both rabbits and chicken according to standard protocols References

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(lv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS /MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (V ) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/027,633

(B) FILING DATE: 01-OCT-1996

(vi ) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: EP 97100583.0

(B) FILING DATE: 16-JAN-1997

(2) INFORMATION FOR SEQ ID NO:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ll) MOLECULE TYPE: peptide

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Gin Arg Arg Ser Arg Thr Asn Phe Thr Leu Glu Gin Leu Asn Glu Leu 1 5 10 15

Glu Arg Leu Phe Asp Glu Thr His Tyr Pro Asp Ala Phe Met Arg Glu 20 25 30 Glu Leu Ser Gin Arg Leu Gly Leu Ser Glu Ala Arg Val Gin Val Trp 35 40 45

Phe Gin Asn Arg Arg Ala Lys Cys Arg Lys Gin Glu 50 55 60

(2) INFORMATION FOR SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 209 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon II: ET93" (v) FRAGMENT TYPE: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GGATTTATGA ATGCAAAGAG AAGCGCGAGG ACGTGAAGTC GGAGGACGAG GACGGGCAGA 60

CCAAGCTGAA ACAGAGGCGC AGCCGCACCA ACTTCACGCT GGAGCAGCTG AACGAGCTCG 120 AGCGACTCTT CGACGAGACC CATTACCCCG ACGCCTTCAT GCGCGAGGAG CTCAGCCAGC 180

GCCTGGGGCT CTCCGAGGCG CGCGTGCAG 209

(2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 368 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon I: G310"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTGATCCACC CGCGCGCACG GGCCGTCCTC TCCGCGCGGG GAGACGCGCG CATCCACCAG 60

CCCCGGCTGC TCGCCAGCCC CGGCCCCAGC CATGGAAGAG CTCACGGCTT TTGTATCCAA 120

GTCTTTTGAC CAGAAAAGCA AGGACGGTAA CGGCGGAGGC GGAGGCGGCG GAGGTAAGAA 180

GGATTCCATT ACGTACCGGG AAGTTTTGGA GAGCGGACTG GCGCGCTCCC GGGAGCTGGG 240

GACGTCGGAT TCCAGCCTCC AGGACATCAC GGAGGGCGGC GGCCACTGCC CGGTGCATTT 300 GTTCAAGGAC CACGTAGACA ATGACAAGGA GAAACTGAAA GAATTCGGCA CCGCGAGAGT 360

GGCAGAAG 368 (2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon III: ET45" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTTTGGTTCC AGAACCGGAG AGCCAAGTGC CGCAAACAAG AGAATCAGAT GCATAAAG 58 (2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 89 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon IV: G108"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCGTCATCTT GGGCACAGCC AACCACCTAG ACGCCTGCCG AGTGGCACCC TACGTCAACA 60 TGGGAGCCTT ACGGATGCCT TTCCAACAG 89 (2) INFORMATION FOR SEQ ID NO: 6:

( ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1166 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon : Va"

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GTCCAGGCTC AGCTGCAGCT GGAAGGCGTG GCCCACGCGC ACCCGCACCT GCACCCGCAC 60

CTGGCGGCGC ACGCGCCCTA CCTGATGTTC CCCCCGCCGC CCTTCGGGCT GCCCATCGCG 120 TCGCTGGCCG AGTCCGCCTC GGCCGCCGCC GTGGTCGCCG CCGCCGCCAA AAGCAACAGC 180

AAGAATTCCA GCATCGCCGA CCTGCGGCTC AAGGCGCGGA AGCACGCGGA GGCCCTGGGG 240

CTCTGACCCG CCGCGCAGCC CCCCGCGCGC CCGGACTCCC GGGCTCCGCG CACCCCGCCT 300

GCACCGCGCG TCCTGCACTC AACCCCGCCT GGAGCTCCTT CCGCGGCCAC CGTGCTCCGG 360

GCACCCCGGG AGCTCCTGCA AGAGGCCTGA GGAGGGAGGC TCCCGGGACC GTCCACGCAC 420 GACCCAGCCA GACCCTCGCG GAGATGGTGC AGAAGGCGGA GCGGGTGAGC GGCCGTGCGT 480

CCAGCCCGGG CCTCTCCAAG GCTGCCCGTG CGTCCTGGGA CCCTGGAGAA GGGTAAACCC 540

CCGCCTGGCT GCGTCTTCCT CTGCTATACC CTATGCATGC GGTTAACTAC ACACGTTTGG 600

AAGATCCTTA GAGTCTATTG AAACTGCAAA GATCCCGGAG CTGGTCTCCG ATGAAAATGC 660

CATTTCTTCG TTGCCAACGA TTTTCTTTAC TACCATGCTC CTTCCTTCAT CCCGAGAGGC 720 TGCGGAACGG GTGTGGATTT GAATGTGGAC TTCGGAATCC CAGGAGGCAG GGGCCGGGCT 780

CTCCTCCACC GCTCCCCCGG AGCCTCCCAG GCAGCAATAA GGAAATAGTT CTCTGGCTGA 840 GGCTGAGGAC GTGAACCGCG GGCTTTGGAA AGGGAGGGGA GGGAGACCCG AACCTCCCAC 900 GTTGGGACTC CCACGTTCCG GGGACCTGAA TGAGGACCGA CTTTATAACT TTTCCAGTGT 960

TTGATTCCCA AATTGGGTCT GGTTTTGTTT TGGATTGGTA TTTTTTTTTT _TTTTTTTTTT 1020

TGCTGTGTTA CAGGATTCAG ACGCAAAAGA CTTGCATAAG AGACGGACGC GTGGTTGCAA 1080 GGTGTCATAC TGATATGCAG CATTAACTTT ACTGACATGG AGTGAAGTGC AATATTATAA 1140

ATATTATAGA TTAAAAAAAA AATAGC 1166 (2) INFORMATION FOR SEQ ID NO: 7:

( ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 625 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "exon Vb"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATGGAGTTTT GCTCTTGTCG CCCAGGCTGG AGTATAATGG CATGATCTCG ACTCACTGCA 60

ACCTCCGCCT CCCGAGTTCA AGCGATTCTC CTGCCTCAGC CTCCCGAGTA GCTGGGATTA 120

CAGGTGCCCA CCACCATGTC AAGATAATGT TTGTATTTTC AGTAGAGATG GGGTTTGACC 180

ATGTTGGCCA GGCTGGTCTC GAACTCCTGA CCTCAGGTGA TCCACCCGCC TTAGCCTCCC 240

AAAGTGCTGG GATGACAGGC GTGAGCCCCT GCGCCCGGCC TTTGTAACTT TATTTTTAAT 300 _{ττττττττττ} TTTTAAGAAA GACAGAGTCT TGCTCTGTCA CCCAGGCTGG AGCACACTGG 360

TGCGATCATA GCTCACTGCA GCCTCAAACT CCTGGGCTCA AGCAATCCTC CCACCTCAGC 420

CTCCTGAGTA GCTGGGACTA CAGGCACCCA CCACCACACC CAGCTAATTT TTTTGATTTT 480

TACTAGAGAC GGGATCTTGC TTTGCTGCTG AGGCTGGTCT TGAGCTCCTG AGCTCCAAAG 540

ATCCTCTCAC CTCCACCTCC CAAAGTGTTA GAATTACAAG CATGAACCAC TGCCCGTGGT 600 CTCCAAAAAA AGGACTGTTA CGTGG 625

(2) INFORMATION FOR SEQ ID NO: 8:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15577 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "HOX93"

(lx) FEATURE: (A) NAME/KEY: exon

(B) LOCATION: 1498..1807

(D) OTHER INFORMATION: /functιon= "part of exon I (G310) " ( ix ) FEATURE :

(A) NAME/KEY: mιsc_feature

(B) LOCATION: 3844..4068 (D) OTHER INFORMATION: /functions "_PET92 region (first part) "

(ix) FEATURE:

(A) NAME/KEY: mιsc_feature (B) LOCATION: 4326..4437

(D) OTHER INFORMATION: /functions "_pET92 region (second part) "

(IX) FEATURE: (A) NAME/KEY: mιsc_feature

(B) LOCATION: 4545..4619

(D) OTHER INFORMATION: /functions "_PET92 region (third part)" (ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 5305..5512

(D) OTHER INFORMATION: /functions "part of exon II (ET93)" (ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 11620..11729

(D) OTHER INFORMATION: /functions "part of exon IV (G108)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CTCTCCCTGT TGTGTCTCTC TTTCTCTCTC TCCATCTCTC TCCGTCTTTC CCCCTCTGTC 60 TCTTTCTCTG TCTCCATCCC TCTGTCTCTC CCTTTCTCTC TGTCTTTCCT TGTCTCTCTC 120

TTTCTCTCTC TCTCTCCATC TCTCTCTCTC CCGGTCTCTC TCTCTCCATC TCCCCGTCTC 180

TCCGTTTCTC TCTCTGCCTC TCCCTGTCTG TCTCTCTCTT TGTGTGTGTT ACACACACCC 240

CAACCCACCG TCACTCATGT CCCCCCACTG CTGTGCCATC TCACACAAGT TCACAGCTCA 300

GCTGTCATCC TGGGTCCCCA GGCCCCGCCG GGGAGGAAGA TGCGCCGTGG GGTTACGGGA 360 GGAAGGGGAC TCCGGGCCTC CTGGTGCCCC ACTTTATTTG CAGAAGGTCC TTGGCAGGAA 420

CCGTGACGCG TTTGGTTTCC AGGACTTGGA AAACGAATTT CAGGTCGCGA TGGCGAGCAC 480

CGGCTTCCCC TGAAGCACAT TCAATAGCGA GAGGCGGGAG GGAGCGAGCA GGAGCATCCC 540

ACCATGAAAA CCAAAAACAC AAGTATTTTT TTCACCCGGT AAATACCCCA GACGCCAGGG 600

TGACAGCGCG GCGCTAAGGG AGGAGGCCTC GCGCCGGGGT CCGCCGGGAT CTGGCGCGGG 660 CGGAAAGAAT ATAGATCTTT ACGAACCGGA TCTCCCGGGG ACCTGGGCTT CTTTCTGCGG 720

GCGCTGGAAA CCCGGGAGGC GGCCCCGGGG ATCCTCGGCC TCCGCCGCCG CCGCCTCCCA 780

AGCGCCCGCG TCCCGGTTTG GGGAGACCCG GCCCCTTCTT CTCACTTTCG GGGATTCTCC 840

AGCCGCGTTC CATCTCACCA ACTCTCCATC CAAGGGCGCG CCGCCACCAA CTTGGAGCTC 900

ATCTTCTCCC AAAATCGTGC GTCCCCGGGG CGCCCGGGTC CCCCCCCTCG CCATCTCAAC 960 CCCGGCGCGA CCCGGGCGCT TCCTGGAAAG ATCCAGGCGC CGGGCTCTGC GCTCCTCCCG 1020

GGAGCGAGGG CGGCCGGACA ACTGGGACCC TCCTCTCTCC AGCCGTGAAC TCCTTGTCTC 1080 TCTGTCTCTC TCTGCAGGAA AACTGGAGTT TGCTTTTCCT CCGGCCACGG AAAGAACGCG 1140

GGTAACCTGT GTGGGGGGCT CGGGCGCCTG CGCCCCCCTC CTGCGCGCGC GCTCTCCCTT 1200

CCAAAAATGG GATCTTTCCC CCTTCGCACC AAGGTGTACG GACGCCAAAC AGTGATGAAA 1260

TGAGAAGAAA GCCAATTGCC GGCCTGGGGG GTGGGGGAGA CACAGCGTCT CTGCGTGCGT 1320 CCGCCGCGGA GCCCGGAGAC CAGTAATTGC ACCAGACAGG CAGCGCATGG GGGGCTGGGC 1380

GAGGTCGCCG CGTATAAATA GTGAGATTTC CAATGGAAAG GCGTAAATAA CAGCGCTGGT 1440

GATCCACCCG CGCGCACGGG CCGTCCTCTC CGCGCGGGGA GACGCGCGCA TCCACCAGCC 1500

CCGGCTGCTC GCCAGCCCCG GCCCCAGCCA TGGAAGAGCT CACGGCTTTT GTATCCAAGT 1560

CTTTTGACCA GAAAAGCAAG GACGGTAACG GCGGAGGCGG AGGCGGCGGA GGTAAGAAGG 1620 ATTCCATTAC GTACCGGGAA GTTTTGGAGA GCGGACTGGC GCGCTCCCGG GAGCTGGGGA 1680

CGTCGGATTC CAGCCTCCAG GACATCACGG AGGGCGGCGG CCACTGCCCG GTGCATTTGT 1740

TCAAGGACCA CGTAGACAAT GACAAGGAGA AACTGAAAGA ATTCGGCACC GCGAGAGTGG 1800

CAGAAGGTAA GTTCCTTTGC GCGCCGGCTC CAGGGGGGCC CTCCTGGGGT TCGGCGCCTC 1860

CTCGCCACGG AGTCGGCCCC GCGCGCCCCT CGCTGTGCAC ATTTGCAGCT CCCGTCTCGC 1920 CAGGGTAAGG CCCGGGCCGT CAGGCTTTGC CTAAGAAAGG AAGGAAGGCA GGAGTGGACC 1980

CGACCGGAGA CGCGGGTGGT GGGTAGCGGG GTGCGGGGGG ACCCAGGGAG GGTCGCAGCG 2040

GGGGCCGCGC GCGTGGGCAC CGACACGGGA AGGTCCCGGG CTGGGGTGGA TCCGGGTGGC 2100

TGTGCCTGAA GCCGTAGGGC CTGAGATGTC TTTTTCATTT TCTTTTTCTT TCCTTTCCTT 2160

TTTTTGTTTG TTTGTTTGTT TGTTTGAGAC AGAGTCTCGC TCTGTCCCCC AGGCTGGAGT 2220 GCAGTGGTGC GATCTCGGCT CACTGCAACC TCCGCCTCCT GGGTTCAAGC GATTCTCCTG 2280

CCTCAGCCTC CCCAGTAGCT GGGATTACAG GCATGCACCA CCACGCCTGG CTAATTTTTG 2340

TGCTTTTAGT AAAGACGGGG ATTCACCATG TTGGCCAGGC TGGTCTCGAA CTCCTGACCT 2400

CAGGTGATCC ACCCGCCTCG GCCTCCCAAA GTGCTGGGAT GACAGGCGTG AGGCACCGCG 2460

CCCGGCCTGG GTCCTGACGG CTTAGGATGT GTGTTTCTGT CTCTGCCTGT CTGCCTTGTA 2520 TTTACGGTCA CCCAGACGCA CAGAGGAGCC GTCTCCACGC GCCTTCCCAG CGCTCAGCGC 2580

CTGCCGGGCC CCCGGAGATC ACGGGAAGAC TCGAGGCTGC GTGGTAGGAG ACGGGAAGGC 2640

CCCGGGTCAG CTCGGTTCTG TTTCNCTTTA AGGAACCCTT CATTATTATT TCATTGTTTT 2700

CCTTTGAACG TCGAGGCTTG ATCTTGGCGA AAGCTGTTGG GTCCATAAAA ACCACTCCCG 2760

TGAGCGGAGG TGGCCGGGAT CTGGATGGGG CGCGAGGGGC CCCGGGGAAG CTGGCGGCTT 2820 CGCGGGCGCG TCCTAAGTCA AGGTTGTCAG AGCGCAGCCG GTTGTGCGCG GCCCGGGGGN 2880

AGCTCCCCTC TGGCCCTTCC TCCTGAGACC TCAGTGGTGG GTCGTCCCGT GGTGGAAATC 2940

GGGGAGTAAG AGGCTCAGAG AGAGGGGCTG GCCCCGGGGA TCTCTGTGCA CACACGACAA 3000

CTGGGCGGCA TACATCTTAA GAATAAAATG GGCTGGCTGT GTCGGGGCAC AGCTGGAGAC 3060 GGCTATGGAC GCCTGTTATG TTTTCATTAC AAAGACGCAG AGAATCTAGC CTCGGCTTTT 3120

GCTGATTCGC AAAGTTGAGG TGCGAGGGTG AATGCCCCAA AGGTAATTCT TCCTAAGACT 3180

CTGGGGCTAC CTGCTCTCCG GGGCCCTGCA TTTGGGGTGT GGAGTGGCCC CGGGAAATAG 3240

CCCTTGTATT CGTAGGAGGC ACCAGGCAGC TTCCCAAGGC CCTGACTTTG TCGAAGCAGA 3300

AAGCTGTGGC TACGGTTTAC AAAGCAGTCC CCGGTTTCTG ACCGTCTAAG AGGCAGGAGC 3360

CCAGCCTGCC TTTGACAGTG AGAGGAGTTC CTCCCTACAC ACTGCTGCGG GCACCCGGCA 3420

CTGTAATTCA TACACAGAGA GTTGGCCTTC CTGGACGCAA GGCTGGGAGC CGCTTGAGGG 3480 CCTGCGTGTA ATTTAAGAGG GTTCGCANGC GCCCGGCGGC CGCTTCTGNT GGGGTTGCTT 3540

TTTGGTTGTC CTTCNGCAAA CACCGTTTTG CTCCTCTNGN AACTCTCTCT TNCTCCCCCN 3600

TGGCCNGTNG GACCCGGGNA NGAGCAAAGT GTCCTCCAGA CCNTTTTGAA ANGTGAGAGG 3660

AAAATAAAGA CCAGGCCAAA NNGACCCAGG GCCACAGGAG AGGAGACAGA GAGTCCCCGT 3720

TACATTTTNC CCCTTGGCTG GGTGCAGAAA GACCCCCGGG CCAGGACTGC CACCCAGGCT 3780 ACTATTTATT CATCAGATCC AAGTTAAATC GAGGTTGGAG GGCAGGGGAG AGTCTGAGGT 3840

TACCGTGGAA GCCTGGAGTT TTTGGGNAAC AGCGTGTCCC CGCCGAGCCT GGGAGCCCGT 3900

GGGTTCTGCA AAGCCTGCGG GTGTTTGAGG ACTTTGAAGA CCAGTTTGTC AGTTGGGCTC 3960

AATTNCCTGG GGTTCAGACT TAGAGAAATG AAGGAGGGAG AGCTGGGGTC GTCTCCAGGA 4020

AACGATTCAC TTGGGGGGAA GGAATGGAGT GTTCTTGCAG GCACATGTCT GTTAGGAGGT 4080 GAAACAGAAT GTGAAATCCA CGTTGGAGTA AGCGTCCAGC GCTGAATGTA GCTCGGGGTG 4140

GGGTGGGAGG GCCCTGGTGT GGATCGTGGA AGGNAAGAAA GACAGAACAG GGTGCTAGTA 4200

TTTACCCCGT TNCCCTGTAG ACACCCTGGA TTTGTCAGCT TTGCAAGCTT CTTGGTTGCA 4260

GCGGCCTTGC CTGTGCCCCT TTGAGACTGT TTCCAGACTA AACTTCCAAA TGTCAGCCCC 4320

TTACCCTTGA CAGCAAGGGA CATCTCATTA GGGCATCGCG TGCTTCTCAT CTGTGNCTCA 4380 GCAGGCCCNG AGATAGGAAN CANGAGGGGC NGTTGGNAGA TGCNCACTTC CACCAGCCCT 4440

GGGNTTGAAG GGGANGCGAN GGGANGACNA CCTTTTANCT TAAACCCCTN GAGCTTGGTN 4500

CAGAGAGGNC TGAATGTCTA AAATGAGGAA GAAAAGGTTT TTCACCTGGA AACGCTTGAG 4560

GGCTGAGTCT TCTGCCCNTT CTGACNTCCC CCAGCAAATA CAGACAGGTC ACCAANCTAC 4620

TGGAGATGAG AAAGTGCCAT TTTTGGCACA CTCTGGTGGG GTAGGTGCCC GACCGCGTGT 4680 GAAAAANGTG GGAANNGGAG AGATTTCTGN CGCACGCGGT TCAGCCCCCA GGCGCGGNTG 4740

GCNGCATTCN AGGNTACTCA GACGCGGTTC TGCTGTTCTG CTGAGAAACA GGCTTCGGGT 4800

AGGGGCTCCT AGCTCCGCCA GATCGCGGAG GGACCCCCAG CCCTCCTGCG CTGCAGCGGT 4860

GGGGATAGCG TCTCTCCGTA GGCCTAGAAT CTGCAACCCG CCCCGGGTCC TCCCCGTGTC 4920

CTTCCCGGGC GTCCCGCCGG GGATCCCACA GTTGGCAGCT CTTCCTCAAA TTCTTTCCCT 4980 TAAAAATAGG ATTTGACACC CCACTCTCCT TAAAAAAAAA AAATAAGAAA AAAAGGTTAG 5040

GTTATGTCAA CAGAGGTGAA GTGGATAATT GAGGAAACGA TTCTGAGATG AGGCCAAGAA 5100 AACAACGCTC GTGCAAAGCC CAGGTTTTTG GGAAAGCAGC GAGTATCCTC CTCGGCTTTT 5160

GCGTTATGGA CCCCACGCAG TTTTTGCGTC AAAGCGCATT GGTTTTCGAG GGCCCCCTTT 5220

CCACCGCGGG ATGCACGAAG GGGTTCGCCA CGTTGCGCAA AACCTCCCCG GCCTCAGCCC 5280

TGTGCCCTCC GCTCCCCACG CAGGGATTTA TGAATGCAAA GAGAAGCGCG AGGACGTGAA 5340 GTCGGAGGAC GAGGACGGGC AGACCAAGCT GAAACAGAGG CGCAGCCGCA CCAACTTCAC 5400

GCTGGAGCAG CTGAACGAGC TCGAGCGACT TTTTGACGAG ACCCATTACC CCGACGCCTT 5460

CATGCGCGAG GAGCTCAGCC AGCGCCTGGG GCTTTCCGAG GCGCGCGTGC AGGTAGGAAC 5520

CCGGGGGCGG GGGCGGGGGG CCCGGAGCCA TCGCCTGGTC CTCGGGAGCG CACAGCACGC 5580

GTACAGCCAC CTGCGCCCGG GCCGCCGCCG TCCCCTTCCC GGAGCGCGGG GAGGTTGGGT 5640 GAGGGACGGG CTGGGGTTCC TGGACTTTTG GAGACGCCTG AGGCCTGTAG GATGGGTTCA 5700

TTGCGTTTGT TTTTCACCAA CAGCAAACAA ATATATATAC ATATATATTA TACAAATAAC 5760

AAATAAATAT ATATGTTATA CAGATGGGTA TATTGTATAT ATTATAGATA TTTGTTCGTC 5820

CTTGGTGCAA AGACACCCGG TGAACCCATA TATTGGCTCC TGACTGCCTT CGGTTCCCCT 5880

GGGATTGGTT ATAGGGGCAA CACATGCAAA CAAAACTTTC CCTGGATTAT ACTTAGGAGA 5940 CGAAGCTACA GATGCGTTTG ATCCAGAGTG TTTTACAAGA TTTTTCATTT AAAAAAAAAT 6000

GTGTCTTTTG GCCCCTGATT CCCCTCCGTC TTCCCGTGTG GCTGCATTGA AAAGGTTTCC 6060

TTAGGATGAA AGGAGAGGGG TGTCCTCTGT CCCTAGGTGG AGAGAAACAG GGTCTTCTCT 6120

TTCCTCCGTT TTTTCACCTA CCGTTTCTAT CTCCCTCCTC CCCTCTCCAG CCCTGTCCTC 6180

TGCTACAAAC CACCCCCTCC TCCCTCCGGC TGTGGGGAGC GCAGGAGCAC GTTGGGCATC 6240 TGGATGAGCG GNAGACTATT AGCGGGGCAC GGGGGCTCCC CGAGGAGCGC GCGAATTCAC 6300

GCTGCCCCAT GAGACCAGGC ACCGGGGGGC GGAGGGGCCT TGGGTGTCCG CAGAGGGACG 6360

GGCGGGCAGA GCCTTCCTCC GCATTCTAAA CATTCACTTA AAGGTATGAG TTTANTTTCA 6420

GGGGTGCTGC TGGGAGAGCC TCCAAATGGC TTCTTCCAGC CCCTGCCTGA CAGTTCAGCT 6480

CCCCTGGAAG GTCAACTCCT CTAGTCCTTT CTCCTGGTTC TGGGCAGGAC AGAAGTGGGG 6540 GGAGGGAGAG AGAGAGAGAG AGAGAGAGAG ACGGTCAGGA TCCCCGGACC CTGGGGAACC 6600

CGTCAAAAAT AAATGAAATT AAGATTGCCG ACCAGAGAGA GAACCGTGAC AAAGCAAACG 6660

GCGTTCAAAG CAAAGAGACG AACTGAAAGC CCGTTCCCGT AGGACTGGTT ATGAGGTCAA 6720

CACATTCAAA CACAGCTTGC TCTGGATTTT GCTGAGCAGA GGAAGATACA GATGCATTTG 6780

ATCCAAAGTG TGTTACATCT TTCATTATAT GTGTGTCTAT ATATATAAAC ATATATAAAT 6840 ATATAAACAT ACATAAATGT ATGTAAATAT ATATAATCTA TATACATATA TAAATATATA 6900

AACACATATA TAATATATAA ATCTATAAAC ATATATAATA TATAAACATA AATATATAAA 6960

CATATATAAT ATATAAATAT ATTAACATAT ATAAAATATG TATAAATATA TATAAACATA 7020

TAAACATATA TAAATATATA AACATATAAA TATATAAACA TATATAAATA TATACAAACA 7080 TATTGTATAT ATATAAATAT ATATAAAAAC ATATATATAC ATATAAAAAT ATATATAAAC 7140

ATATATACAT ATAAAGAAAT ATATATAAAC ATATATACAT ATAAATATAC ATATATAAAC 7200

ATATATATAC ATAAAATATA TATAAACATA TATACATATA AAAATATATA TATATTAACA 7260

TATATATACA TATAAAAATA TATATATTAA CATATATATA CATATAAAAA TATATATATA 7320

TTTTTGGCCC CTGATTCCCT TCGGTTCCTG TGGGATGGGT GATTGAGTCA ACACATTCAA 7380

ACACAACTTT TCCATCGATG TTGCTTAGGA GATGAGGATA CAGATGCGTT TGATGGAGAG 7440

GGTTTTACAA GCTCTTTCAT TTAAATATAT ATATATATAT ATATATATTT TTTGGCTCCT 7500 GATTCTCTTC CGTCTTCCCA TGTGGCTGCA TTTTAAAAGG CTTCCCTAAG ATCGTTACGA 7560

TTAAATCAAC CCTCCCCAGG CATCTTTACC GAGGGCTGTG GTCCCCAAAG CGATACAGCC 7620

CAGGAGGGAG AGAGGCTTTG GTGACTTGGA GGAAGGACTG TGTCCCTCCT TAGGGCGTCT 7680

GTGGCCTCAG TGAGGGAAGG AAGCTGCATC AGACAGGGGT TTCCTCGCTG TCCACCCCTC 7740

TGGCAGAAGA TGGATTGGGC TGCCCCGNTA TAAATTAATG AAAAGATTAA AGTTTCGCTA 7800 AAGGGGACAT CGAGTTTATG TGTCATCTCC TGGTGNTCTG TGTGCCNTGG GATNCTGCAA 7860

TATATCCCAN NGCCCTTGAT GNNNTACTGT TTNCTATAAA AANNTAAATN TACTTGTNNA 7920

ATTTAANTTC CNNNACACTA TTTNCTTTCC NNGTNAGTCT NATTANCCGA NCGAGAGCAN 7980

CGNTTAGTTN CAGCTNGCGG AAAATTGGTT GTGGGGTGTG TGCGGACCCC NGAGNAACGC 8040

CCNNTAAAAT NAAAGACAAA NTCNGGGGAC AAGNCTNGGG GGTTATCGNN ATTGCNNAGG 8100 GGTCGNCATG AAAANTTTAA CGACGGTAAA TAATAATAAA AANNCAAACA TGGGAATGNC 8160

AATAAAAGAC ATAATTCTCC NNATCGCCGC GGGGGGAAAG GATCCTATAG TAAAGGCGAG 8220

TGCGCTTTGA GGGGTCATAA AAATCAATTA GTTCCAACAC CCACGTCCCG CGTTGAGGGG 8280

ACGGGGACGA GCAGGGACAG AAAAAGAAAC CATATTTGAA TCCCATCTCT CTGTGAATTC 8340

TTGGGTCACA TGCGTCTCAG TACAGCCCGT CCCGTGCTGT GACCGGATAG AGTTTCAATT 8400 TACTGTGGAA ATTTGCTGTA AATAAATTGA GCATCCGATA GAAGCTGTTG CTGATTAACC 8460

TTTTATTTTT AGCGTGGCCC TGCAAAGTCG TATCACCCAG CTGTCAGGCT TCTAATCGAA 8520

AGTTATGAGA CCACGGTGAG GGGCAGGCGG TAATTTAATT ACAACAAATA TCTTTGGGTT 8580

TATGGCGCAG AGCTAAATTA AATGTCATTA TTCACTGTCT GTNAATGGNA AATCAAAANN 8640

GGAAATCGCA NTTACGGNCA TTTGGGNNAA ANGAAAGCGG GGNAGTGCTC TTTAATNGAA 8700 NNGAAATAAC TGTCTTAAGC AGTGTCACAC ACTTCACTTA CCATATTCGN GGCCTNAATT 8760

GGAANNTGGA TCGTNNGAAT CACTCCNAAG ACTNGATTTA TTANGCGCTT CACGNCAGCN 8820

NGGCNTAATT CATCNACTTN NGTATTCTTC ATCNNNNATT TTTTTTTTTC CTCTCNNGCC 8880

GTGTTNNGAA GGGAGAGTGA ATGAGGCTTT CCACGTTTCA GGAGGATTTT CTTTTTTGAA 8940

AAATGCCCTT CCAGAGGCTT TTGGGTGGCT GGCTTGCTTT CTGGGCCCTG GAGGANGACA 9000 GGCGGANGAG TCCAGGTGGG CATGGAGAGG CACAGTGGCA GGTCACCTGG ATGGTCAGTG 9060

GAGGTGGAGG TCTGAAGGCG CCAGCTTTGG AAATTATTGG TGAATTTCGA TGTCAGCACC 9120 AGGNCAGGGG CCTTTTTGGC GGGGGTGTGA GGGANGGATG ANCTTTGCTG GGAAANNCAG 9180

GATCAGGTTC TCCAGGCGCA CTGCAGCCCG GTAGGACCCA CTTTGGAAAT GAAAAGCCAG 9240

TTNCCGAAAG CTGGGCTGGA AGCTTCCGTG TTGGGTTCAA GAGCAAGTTC ACGTTGCGCT 9300

GTGTAGACTC CTGGCTGCTC CCAAACTCTG AGGGTTTTCT GAGGTTCCCT TCATAGGGGC 9360 ACCGGCCCTG GGCCATGCAC AGTGCGTAAG GGTGGCTGTG GGCCGAGGGA CCCAGCACGT 9420

GTTTTGCCCA CAACAGCCGG AGTGACTGGT TCACTCACCG CCTTGGCGGA GGACGCCTGT 9480

TCTCTGGACG AATCATTTCT CTTGGGTGGT GACTGCCTTG TGGGTCAAGG TGCAGGTTTT 9540

CTGCCACAGA AAACCTGTTA GGAGGAATTA AGCGACTAAG ACTGTCAGGG AGGTGGTGGT 9600

GGGGGANGAG GNAGGGGGTG GTGTCCAGAT TACCAGGCAT AGGCTAAACT GCCTGCACTC 9660 TCCAGCTGGT CTGTCTGTGG AGGAGGGGAT TGTCAATACT GGGAGAGCAG AGGAGGCTCG 9720

TAGGAGGTGA GAGGGGGTGG AATTTGCATG CAAATCTTCA CATGAGGCCT GTGTGAATTT 9780

CTCCAGCCTC CTGAGGGTCC CCTGCGCTAT TGCACTCAAC TTCTTGATAG TTTACCCCAA 9840

GACTCAGAAG TCCTTAGAGG GGCAGAATGC CCCCACCACA AAGCCTGCTA TCCTTGGGCG 9900

TCCTCAGGAC CCTTGGTCAT GAATGGGACC CTTTCATGTA TGGGGACCCT TGGTAATATG 9960 AATGGGACGC CTTCAGCTCC CCAGGGCTTC CGAGGAGGCC GAGAAGGGCA AAGACACTTC 10020

CGAGGAGGCC GAGAAGGGCA AAGACATTTT CTGGGCTTGG TGTGTCAAGA GCTAGATTGG 10080

AGAAGGGGCT GGATTTGGAA CTCTTTAGCC ATCAGCTCAC CCTCTCCGTT TGTGGCTAAA 10140

GTCTGAAGGT GGAAACTTCG GTTCTCCTAC AGGGTCTACA GGAGTTGGGG GGCGGGGCGC 10200

CCACACAGAA CGCTGGAAAG TTCGACAGTC CACTTCCACT GGCTCGGAAC TCACTTTTTC 10260 ACCTTAAGTT CATCAGCGGT AACGCATAGG TCTCACTTAG GCAGGGCACG GATGATTTAA 10320

CAATTTCTAC TTCTAGGTCA GGTGCGGTGG CTCACACCTC TAATCCCAGC ACTTTGGGAG 10380

GCCCAGGAGG GTGGATCGCT TGAGGTCAGG AGTTTGAGAC CAGCCTGGCC AACATGGTGA 10440

AACCCCGTCT CTACTAAAAT ACGAAAATTA GCCAGGCATG GTGGTGAGCA CCTGTAATTC 10500

CAGCTACTCG GGAGGCTGAG GCAGGAGAAT CGCTTGAACC TGGGAGGTGG ACGTTGCAGT 10560 GAGGTGAGAT CACACCACTG CACTCCAGCC TGGATGAGAG AGCAAGACTC TGTCTCAAAA 10620

ACAAAATAAA ACAAAAACAA AACAAAAATC AAAAAAGAAA ACCCAATTTC CAGTTCTAGG 10680

CCAGGTGCAG TGGCTCACGC CTGTCATCCC AGCACTTTGG GAGGCCCAGG AGGGTGGATC 10740

GCTTGAGGTC AGGAGTTCGA GACCAGCCTG GCCAACATGG TGAAACCCCA TCTTTACTAA 10800

AAATACAAAC GTTAGCTGGG TGTGGTGGTG TGCGCCTGTA ATCCCAGCTA CTCGGGAAGC 10860 TGAGGCTGGA GAATTGCTTG AATCTGGGAG GTGGAGGTTG CAGGGAGGCG AGATAGTGCC 10920

ACTGCAGTCC AGCCTGGACC AGAGAGCAAG ACTCCGTCTC AAAAACAAAA GAAAGCAAAA 10980

ACAAAAAACA AGAGACCAGC CTGGCCAACA TGGTGAAACC GCGTCTTTAC TAAAATACAA 11040

AATTAGCCGG GCATGGTGGT GGGCACCTGT AGTCCCAGCT ACTCGGGAGG CTGAGGCAGG 11100 AGAATGGCTT GAACCTGGGA GGTGGAGCTT GCAGTGAGCC GAGATAGTGC CACTGCACTC 11160

CAGCCTGGGC GACAGAGCGA GACTTGATTT CAGAACCACC ACCACCACAA CAAAACAAAA 11220

CAAAAAATCC AAAAAAACCC CAATTTCCAG TACTAGGTAG TCAGTGATGC AGGGCTGGAG 11280

ACAGAGGGGC GGTAAGTGTC TGGGCGCCCA CCATCAGTCA CCTCCCAGCT CCCANGAGGT 11340

GCAAAGTGCT TGGTTCAGCC TCATGGGAAG GATGCTCCCT GGGGAGGCTG GGCTGGGTTC 11400

ACAGGGCTCT TCACATCTCT CTCTGCTTCT NCCCCAAGGT TTGGTTNCCA GAACCGGAGA 11460

GCCAAGTGCC GNCAAACAAG AGAATCAGAT GCATAAAGGT GGGTGTCGGG ACTGGGGGGA 11520 CCTGAAGCTG GGGGATCCTG CTCCAGGAGG GATGGGGTCG ACAAGGTGCT GGCTACACCC 11580

AGGACCACCA CACTGACACC TGCTCCCTTT GGACACAGGC GTCATCTTGG GCACAGCCAA 11640

CCACCTAGAC GCCTGCCNGA GTGGCACCCT ACGTCAACAT GGGAGCCTTA CGGATGCCTT 11700

TCCAACAGGT AGCTCACTTT TTCTTCCTCT GNAAGATCCC TAGGGACCTG CTGCTCCCTT 11760

CCCCTTTCCC CTATTTGCTG CCGCATCCTG ACACTCCTAG TCCCTCCCTG CCCCTGCAGA 11820 CTTCTCAGCT GGCCCTTAGA AAAAAAGCCT CTTTTCCGAG GAGGCATTTA CAGGCACCTT 11880

GGCACCTATG AAATCAGGCT GGGCCAGGCG GGGTGGCTCA CACCTGTCAT CCCAGCACTT 11940

TGGGAGGCCA AGGTTAGGAG TTTGAGACCA GCCTGGACAA CATAGCAAAA GCCTGTCTCT 12000

ACTAAAAATA CAAAAAAAAA TTAACAGGGA GTGGTGGTGG GCACCTGTAA TCCCAGCTAC 12060

TTGGGAGGCT GAGGCAGGAG AATCACTTGA ACCCGGGAGG CCGAGGTTGC GGTGAGCCGA 12120 GATCGTGCCA TTGCACTCCA GGCTGGGCGA CAGAGTGAGA CTCTGTCTCA AAAAATAAAT 12180

AAATAAATAA ATGTAAAAAA ATAAAAATAG GTCGGGCACG GTGGCTCACG TCTGTAATCC 12240

CAGCACTTTG GAAGGCCGAG GTGGGTGGAT GACAGGGTCA AGAGATTGAG ACCATCCTGG 12300

CCAACATGGC AAAATGCCGT CTCTACTAAA AAATACAAAA ATTAGGCGGG CGTGGTGGCG 12360

GGTGCCTGTA ATCCCAGCTA CTCGGGAGGC TGAGGCAGGA GAATCGGTTG AACCCGGGAT 12420 GCGGAGGTTG CAGTGAGCGG AGATCACATC ACTGCACTCC AGGCTGGGCA ACAAGAGCGA 12480

AACTGCGTCT TACAATAAAT AAATAGATAA ATAAATAAAC AAATAAACTT TACTTTAGAA 12540

ACAAATCCCT GTCCGTGTTT GTCTTTTCAC CTGTCCTGCA GGGAAAACAA AACATAAAAT 12600

GTCAAGGCAA ATAGTAGTGA TTTCATTCCG GGAAAAAGAA AGTGGATGTT TGCCTTCACC 12660

CTTTCTCGTC CTTCCTCTGG TGCTCCTCAN GGCCCANGGG NAGAGGGTGG AAAGTNCAGA 12720 GGAAGAAAGA CGGGGCTGGG GGGGGGGGTC CGTGGGGACC CAGGCAGGCA TGTTCCCNAT 12780

TTCCNTGTCT TCACNTTCAA AGNAGGGGCC CCTCGNCTCT GGAATGAGGC CTACGGTTTC 12840

CTTTCCCNGA AGAGTTNCCC CTTTGTGAGC TTACGGCTTC GGAGTGAACC TCGGTGCAAC 12900

CTGTTATTAA AACACACAGA GGCTAATGCC AGCAAAAACA CGCCCCCCGC TCCTGGTTTC 12960

AGAGGGAAGA AAAAAATTCA TAAGCACGGC CATGCTTTTC TAATAAAAAT TCATTAAATA 13020 ATCGTTATAA GGGATGAAGC CGGGAGGGGA GAGGAGAGGA ACACAATCAA GAGACTTTCT 13080

TTGAACTTTT TCTCCCTGCT TCAAATACAA AGCAATCTTC TGTGGGCCTG GGCCTGGGGG 13140 GTTTCCCCCT TTCTCTGCAG CCCATTGGGA GGAAGAAAAT GCTTCCCTGA ANGTTGCTGC 13200

AAAATTGTTT CTGTTTTTCT TTTCTTTTTC TTTTTTTTTT TTTTTTGAGA CGGAGTCTCG 13260

CTCTGTCACC AGGCTGGAGT GCAATGGTAT GATCTCAGCT CACTGCAACC TCCACGTTCC 13320

TGTTTCAAGT CATTCTCCTG CCTCAGCCTC CTGAGTAGCT GGGACTACAG GCGCCCGCCA 13380 CCACGCCCGG CTAGTGTTTG TATTTTTAGA AAAGACAGGG TTTCCCCATG TTGGCCAGGC 13440

TGGTCTTGAA CTCCTGTCCT CAAGTGATCT GCCTGCCTCG GCCTCCCAAA GTGCTGTGTT 13500

TCTGTTTTTC TTTCCCCGCT TTCTTAGGAG GCCATCGGGA AGAATAAAAT GCTTTCCTTG 13560

AAGTTGATGC AAAATTGTTT CTGTTTTTCT TTTCTCTTTT CTTTCTTTTT GAGATGGAGT 13620

CTCGCTCTTT CACCCAGGCT GGAGGGCAGT GGCGCGACCT CGGCTCACTG CAACCTCCGC 13680 CTCCCGGGTT CAAGCGATTC TCCTGCCTCA GCCTCCGGAG TAGCTGGGAT TACAGGCACC 13740

TGCCACTATG CCTGGCTAAT TTTATTATTT TTAGTAGAGA CGGGGTTTCA CCATGTTGGC 13800

CAGGCTGGTC TCAAACTCCT GACCTCAGGT GATCCGCCCG CCTCGCCTCC CAAAGTGATG 13860

GGATGANCAG GNCATNGAGC NCACCGTGCC CGGCCCTCTA ACTCTTTACC AGACATAAAG 13920

TCTCCNNTTC CCCTTTCTAA ATGTATATAT TGTGTTTTTA AAAGTTAACA GCAGGGATCC 13980 CACCTCATTN CCCCGCTNCT CTCCCCAAGA CCTGTCCTGC ACGTTGCACA CAGCAGGTGT 14040

GCCCTGGACA TATCCCAAAC CCACGCTGAA AGAAAGAGGG TCTCACTACA CGTATGATAT 14100

CTGTGNATCC TTTAAACATC TCCGTGGCTT CCAGGCAACA CAGCCATAAA TAGGAATCTC 14160

ATGTCTGACA TGATACCGGG ACCATGTATG GGNAAATTCT GGGTGTGAAG TTCCAGCTAC 14220

CCCCGCAGAG GCANCCATTG CATACCCTCC AGAAACTCCC CTGCCGTTNC AAGCCAAAGA 14280 CACAACACAA ACAGCNTCCG AGAGAGGGTG TCATTGAAAA TCAATACCAT CATAAGAGCA 14340

CACAGCACCG TCTTTCTCTT CTGCCCGTTG ATACACAATT ATGAGCAATT TGCTAACACT 14400

GACAACTCGT GGCAAGAACA GGTCGTGTTG ATACGGTTGC CTCGTGAGGA CCCATCTGTC 14460

TTCTGGGGTC TTGCCTGGAA CGGAGATCGG AGTTCAGGGT GGCTAATAGA ATCATTACTC 14520

ACCTAGGGAC ACAGAATNAT GAGGGTTACC CCCAGTTAAG TGCATACAGT CAAACGGACG 14580 GCTGCTCTGG AAGGTACAGT GACGTGAACA GCTTTTATGA AATGCCTAGA TCTGGACCTT 14640

CCATACCTGA GCCACCGTTC CAAAGCACTG GGCGTTTTTC AGATACTTTC ATGAGAAATG 14700

TTGTCAACAC CGCAAGTTTG CAGTACACAG TCTGAAAGAT ATTCTTGTAT ATGTAGATGT 14760

CTGTAGATGC CCTGAAGGTG TGTAGACTTT AGACACCCAG AAGGTGTGTA GATGTCTGTA 14820

GACACCTTCT ATGTGTGTAG ATGTCTGTAG ACGCCCTGCA GGTGTGTAGA TATATCTAGA 14880 TGGTCTGCCT GTGTATGATA CAGGCTAAAA AGACATTTGT GGTGGACACT AGTTGATTAT 14940

TTAGGACTAT GAGATGGGAA AGGAAGNAGC AACCAGCAGT GAAAGGCATG TGGTGGGTGG 15000

GGGGTTGGCA TTGCAGTGGG GTCCTCNTGA NGCAGGTGAC ACCCACTATA GGGCTGCCCT 15060

TGGNATGGAC GCTTTGTNGA AGCTGTTTGA TTTCACCACA CCAAGCCTGG AGGCACGGAC 15120 ATTCCAGGAT GGTGAGGAGT CTGCAAAGGA GGAGATTGGA GGAGGTGCAA TATCCCTAGA 15180

GTACGAGAGA TGAGATAGGA GAGCTGTATA AATAGCACTA CCAGCCGGAT GCGGTGGCTC 15240 ACGCCTGTCA TCCCAGCACT TTAGGAGGCT GAGGCAGGCG GATCACCTGA GGTCAGGAGT 15300

TCCAGAACAG CCTGGCCAAC ACAATGAAAC CCCATCTTTA CTAAAAATAC AAGATTAGCT 15360

GGGCACGGTG TCTCACGCCT GTCATCCCTG CACTTTGGGA GGTCGAGGTG CGCAGATCAT 15420

GAGGTCAGTT TGGCCAACGC GGCGAAACCC CGTCTCTACT AAAAATACAA AAAAGTAGCC 15480

GGGCGTGGTG GTGGGCACCT GTAGTCCCAG CTACTAGGGA GGCTGAGGCA GGAGAATCGC 15540 TTGAACCCGG ATGCGGACAT TGCAGTGAGC CGAGATC 15577

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 753 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii ) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "ET92 gene segment"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CGTGGAAGCC TGGAGTTTTT GGGAACAGCG TGTCCCCGCC GAGCCTGGGA GCCCGTGGGT 60 TCTGCAAAGC CTGCGGGTGT TTGAGGACTT TGAAGACCAG TTTGTCAGTT GGGCTCAATT 120

CCTGGGGTTC AGACTTAGAG AAATGAAGGA GGGAGAGCTG GGGTCGTCTC CAGGAAACGA 180

TTCACTTGGG GGGAAGGAAT GGAGTGTTCT TGCAGGCACA TGTCTGTTAG GAGGTGAAAC 240

AGAATGTGAA ATCCACGTTG GAGTAAGCGT CCAGCGCTGA ATGTAGCTCG GGGTGGGGTG 300

GGAGGGCCCT GGTGTGGATC GTGGAAGGAA GAAAGACAGA ACAGGGTGCT AGTATTTACC 360 CCGTTCCCTG TAGACACCCT GGATTTGTCA GCTTTGCAAG CTTCTTGGTT GCAGCGGCCT 420

TGCCTGTGCC CCTTTGAGAC TGTTTCCAGA CTAAACTTCC AAATGTCAGC CCCTTACCCT 480

TGACAGCAAG GGACATCTCA TTAGGGCATC GCGTGCTTCT CATCTGTGCT CAGCAGGCCC 540

GAGATAGGAA CAGAGGGGCG TTGGAGATGC CACTTCCACC AGCCCTGGGT TGAAGGGGAG 600

CGAGGGAGAC ACCTTTTACT TAAACCCCTG AGCTTGGTCA GAGAGGCTGA ATGTCTAAAA 660 TGAGGAAGAA AAGGTTTTTC ACCTGGAAAC GCTTGAGGGC TGAGTCTTCT GCCCTTCTGA 720

CTCCCCCAGC AAATACAGAC AGGTCACCAA CTA 753 (2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1890 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "SHOXa"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 91..968

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GTGATCCACC CGCCGCACGG GCCGTCCTCT CCGCGCGGGG AGACGCGCGC ATCCACCAGC 60

CCCGGCTGCT CGCCAGCCCC GGCCCCAGCC ATG GAA GAG CTC ACG GCT TTT GTA 114 Met Glu Glu Leu Thr Ala Phe Val

1 5

TCC AAG TCT TTT GAC CAG AAA AGC AAG GAC GGT AAC GGC GGA GGC GGA 162 Ser Lys Ser Phe Asp Gin Lys Ser Lys Asp Gly Asn Gly Gly Gly Gly 10 15 20

GGC GGC GGA GGT AAG AAG GAT TCC ATT ACG TAC CGG GAA GTT TTG GAG 210 Gly Gly Gly Gly Lys Lys Asp Ser He Thr Tyr Arg Glu Val Leu Glu 25 30 35 40

AGC GGA CTG GCG CGC TCC CGG GAG CTG GGG ACG TCG GAT TCC AGC CTC 258 Ser Gly Leu Ala Arg Ser Arg Glu Leu Gly Thr Ser Asp Ser Ser Leu 45 50 55 CAG GAC ATC ACG GAG GGC GGC GGC CAC TGC CCG GTG CAT TTG TTC AAG 306

Gin Asp He Thr Glu Gly Gly Gly His Cys Pro Val His Leu Phe Lys 60 65 70

GAC CAC GTA GAC AAT GAC AAG GAG AAA CTG AAA GAA TTC GGC ACC GCG 354 Asp H s Val Asp Asn Asp Lys Glu Lys Leu Lys Glu Phe Gly Thr Ala 75 80 85

AGA GTG GCA GAA GGG ATT TAT GAA TGC AAA GAG AAG CGC GAG GAC GTG 402

Arg Val Ala Glu Gly He Tyr Glu Cys Lys Glu Lys Arg Glu Asp Val 90 95 100

AAG TCG GAG GAC GAG GAC GGG CAG ACC AAG CTG AAA CAG AGG CGC AGC 450

Lys Ser Glu Asp Glu Asp Gly Gin Thr Lys Leu Lys Gin Arg Arg Ser

105 110 115 120

CGC ACC AAC TTC ACG CTG GAG CAG CTG AAC GAG CTC GAG CGA CTC TTC 498 Arg Thr Asn Phe Thr Leu Glu Gin Leu Asn Glu Leu Glu Arg Leu Phe 125 130 135 GAC GAG ACC CAT TAC CCC GAC GCC TTC ATG CGC GAG GAG CTC AGC CAG 546 Asp Glu Thr His Tyr Pro Asp Ala Phe Met Arg Glu Glu Leu Ser Gin 140 145 150

CGC CTG GGG CTC TCC GAG GCG CGC GTG CAG GTT TGG TTC CAG AAC CGG 594 Arg Leu Gly Leu Ser Glu Ala Arg Val Gin Val Trp Phe Gin Asn Arg 155 160 165

AGA GCC AAG TGC CGC AAA CAA GAG AAT CAG ATG CAT AAA GGC GTC ATC 642 Arg Ala Lys Cys Arg Lys Gin Glu Asn Gin Met His Lys Gly Val He 170 175 180

TTG GGC ACA GCC AAC CAC CTA GAC GCC TGC CGA GTG GCA CCC TAC GTC 690 Leu Gly Thr Ala Asn His Leu Asp Ala Cys Arg Val Ala Pro Tyr Val 185 190 195 200

AAC ATG GGA GCC TTA CGG ATG CCT TTC CAA CAG GTC CAG GCT CAG CTG 738 Asn Met Gly Ala Leu Arg Met Pro Phe Gin Gin Val Gin Ala Gin Leu 205 210 215

CAG CTG GAA GGC GTG GCC CAC GCG CAC CCG CAC CTG CAC CCG CAC CTG 786 Gin Leu Glu Gly Val Ala His Ala His Pro His Leu His Pro His Leu 220 225 230

GCG GCG CAC GCG CCC TAC CTG ATG TTC CCC CCG CCG CCC TTC GGG CTG 834 Ala Ala His Ala Pro Tyr Leu Met Phe Pro Pro Pro Pro Phe Gly Leu 235 240 245

CCC ATC GCG TCG CTG GCC GAG TCC GCC TCG GCC GCC GCC GTG GTC GCC 882 Pro He Ala Ser Leu Ala Glu Ser Ala Ser Ala Ala Ala Val Val Ala 250 255 260 GCC GCC GCC AAA AGC AAC AGC AAG AAT TCC AGC ATC GCC GAC CTG CGG 930 Ala Ala Ala Lys Ser Asn Ser Lys Asn Ser Ser He Ala Asp Leu Arg 265 270 275 280

CTC AAG GCG CGG AAG CAC GCG GAG GCC CTG GGG CTC TG ACCCGCCGCG 978 Leu Lys Ala Arg Lys His Ala Glu Ala Leu Gly Leu

285 290

CAGCCCCCCG CGCGCCCGGA CTCCCGGGCT CCGCGCACCC CGCCTGCACC GCGCGTCCTG 1038 CACTCAACCC CGCCTGGAGC TCCTTCCGCG GCCACCGTGC TCCGGGCACC CCGGGAGCTC 1098

CTGCAAGAGG CCTGAGGAGG GAGGCTCCCG GGACCGTCCA CGCACGACCC AGCCAGACCC 1158

TCGCGGAGAT GGTGCAGAAG GCGGAGCGGG TGAGCGGCCG TGCGTCCAGC CCGGGCCTCT 1218

CCAAGGCTGC CCGTGCGTCC TGGGACCCTG GAGAAGGGTA AACCCCCGCC TGGCTGCGTC 1278

TTCCTCTGCT ATACCCTATG CATGCGGTTA ACTACACACG TTTGGAAGAT CCTTAGAGTC 1338 TATTGAAACT GCAAAGATCC CGGAGCTGGT CTCCGATGAA AATGCCATTT CTTCGTTGCC 1398

AACGATTTTC TTTACTACCA TGCTCCTTCC TTCATCCCGA GAGGCTGCGG AACGGGTGTG 1458

GATTTGAATG TGGACTTCGG AATCCCAGGA GGCAGGGGCC GGGCTCTCCT CCACCGCTCC 1518

CCCGGAGCCT CCCAGGCAGC AATAAGGAAA TAGTTCTCTG GCTGAGGCTG AGGACGTGAA 1578

CCGCGGGCTT TGGAAAGGGA GGGGAGGGAG ACCCGAACCT CCCACGTTGG GACTCCCACG 1638 TTCCGGGGAC CTGAATGAGG ACCGACTTTA TAACTTTTCC AGTGTTTGAT TCCCAAATTG 1698

GGTCTGGTTT TGTTTTGGAT TGGTATTTTT TTTTTTTTTT TTTTTTGCTG TGTTACAGGA 1758

TTCAGACGCA AAAGACTTGC ATAAGAGACG GACGCGTGGT TGCAAGGTGT CATACTGATA 1818

TGCAGCATTA ACTTTACTGA CATGGAGTGA AGTGCAATAT TATAAATATT ATAGATTAAA 1878

AAAAAAATAG CA 1890

(2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 292 ammo acids (B) TYPE: ammo acid

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(x ) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Glu Glu Leu Thr Ala Phe Val Ser Lys Ser Phe Asp Gin Lys Ser 1 5 10 15 Lys Asp Gly Asn Gly Gly Gly Gly Gly Gly Gly Gly Lys Lys Asp Ser 20 25 30

He Thr Tyr Arg Glu Val Leu Glu Ser Gly Leu Ala Arg Ser Arg Glu 35 40 45

Leu Gly Thr Ser Asp Ser Ser Leu Gin Asp He Thr Glu Gly Gly Gly 50 55 60

His Cys Pro Val His Leu Phe Lys Asp His Val Asp Asn Asp Lys Glu 65 70 75 80

Lys Leu Lys Glu Phe Gly Thr Ala Arg Val Ala Glu Gly He Tyr Glu 85 90 95

Cys Lys Glu Lys Arg Glu Asp Val Lys Ser Glu Asp Glu Asp Gly Gin 100 105 110 Thr Lys Leu Lys Gin Arg Arg Ser Arg Thr Asn Phe Thr Leu Glu Gin 115 120 125

Leu Asn Glu Leu Glu Arg Leu Phe Asp Glu Thr His Tyr Pro Asp Ala 130 135 140

Phe Met Arg Glu Glu Leu Ser Gin Arg Leu Gly Leu Ser Glu Ala Arg

145 150 155 160

Val Gin Val Trp Phe Gin Asn Arg Arg Ala Lys Cys Arg Lys Gin Glu 165 170 175

Asn Gin Met His Lys Gly Val He Leu Gly Thr Ala Asn His Leu Asp 180 185 190 Ala Cys Arg Val Ala Pro Tyr Val Asn Met Gly Ala Leu Arg Met Pro 195 200 205

Phe Gin Gin Val Gin Ala Gin Leu Gin Leu Glu Gly Val Ala His Ala 210 215 220

His Pro His Leu His Pro His Leu Ala Ala His Ala Pro Tyr Leu Met

225 230 235 240

Phe Pro Pro Pro Pro Phe Gly Leu Pro He Ala Ser Leu Ala Glu Ser 245 250 255

Ala Ser Ala Ala Ala Val Val Ala Ala Ala Ala Lys Ser Asn Ser Lys 260 265 270 Asn Ser Ser He Ala Asp Leu Arg Leu Lys Ala Arg Lys His Ala Glu 275 280 285

Ala Leu Gly Leu 290

(2) INFORMATION FOR SEQ ID NO: 12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1354 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "SHOXb" ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 91..768

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GTGATCCACC CGCCGCACGG GCCGTCCTCT CCGCGCGGGG AGACGCGCGC ATCCACCAGC 60 CCCGGCTGCT CGCCAGCCCC GGCCCCAGCC ATG GAA GAG CTC ACG GCT TTT GTA 114

Met Glu Glu Leu Thr Ala Phe Val 295 300

TCC AAG TCT TTT GAC CAG AAA AGC AAG GAC GGT AAC GGC GGA GGC GGA 162 Ser Lys Ser Phe Asp Gin Lys Ser Lys Asp Gly Asn Gly Gly Gly Gly

305 310 315

GGC GGC GGA GGT AAG AAG GAT TCC ATT ACG TAC CGG GAA GTT TTG GAG 210 Gly Gly Gly Gly Lys Lys Asp Ser He Thr Tyr Arg Glu Val Leu Glu 320 325 330

AGC GGA CTG GCG CGC TCC CGG GAG CTG GGG ACG TCG GAT TCC AGC CTC 258 Ser Gly Leu Ala Arg Ser Arg Glu Leu Gly Thr Ser Asp Ser Ser Leu 335 340 345

CAG GAC ATC ACG GAG GGC GGC GGC CAC TGC CCG GTG CAT TTG TTC AAG 306 Gin Asp He Thr Glu Gly Gly Gly His Cys Pro Val His Leu Phe Lys 350 355 360 GAC CAC GTA GAC AAT GAC AAG GAG AAA CTG AAA GAA TTC GGC ACC GCG 354 Asp His Val Asp Asn Asp Lys Glu Lys Leu Lys Glu Phe Gly Thr Ala 365 370 375 380

AGA GTG GCA GAA GGG ATT TAT GAA TGC AAA GAG AAG CGC GAG GAC GTG 402 Arg Val Ala Glu Gly He Tyr Glu Cys Lys Glu Lys Arg Glu Asp Val

385 390 395

AAG TCG GAG GAC GAG GAC GGG CAG ACC AAG CTG AAA CAG AGG CGC AGC 450 Lys Ser Glu Asp Glu Asp Gly Gin Thr Lys Leu Lys Gin Arg Arg Ser 400 405 410

CGC ACC AAC TTC ACG CTG GAG CAG CTG AAC GAG CTC GAG CGA CTC TTC 498 Arg Thr Asn Phe Thr Leu Glu Gin Leu Asn Glu Leu Glu Arg Leu Phe 415 420 425

GAC GAG ACC CAT TAC CCC GAC GCC TTC ATG CGC GAG GAG CTC AGC CAG 546 Asp Glu Thr His Tyr Pro Asp Ala Phe Met Arg Glu Glu Leu Ser Gin 430 435 440 CGC CTG GGG CTC TCC GAG GCG CGC GTG CAG GTT TGG TTC CAG AAC CGG 594 Arg Leu Gly Leu Ser Glu Ala Arg Val Gin Val Trp Phe Gin Asn Arg 445 450 455 460

AGA GCC AAG TGC CGC AAA CAA GAG AAT CAG ATG CAT AAA GGC GTC ATC 642 Arg Ala Lys Cys Arg Lys Gin Glu Asn Gin Met His Lys Gly Val He

465 470 475

TTG GGC ACA GCC AAC CAC CTA GAC GCC TGC CGA GTG GCA CCC TAC GTC 690 Leu Gly Thr Ala Asn His Leu Asp Ala Cys Arg Val Ala Pro Tyr Val 480 485 490

AAC ATG GGA GCC TTA CGG ATG CCT TTC CAA CAG ATG GAG TTT TGC TCT 738 Asn Met Gly Ala Leu Arg Met Pro Phe Gin Gin Met Glu Phe Cys Ser 495 500 505

TGT CGC CCA GGC TGG AGT ATA ATG GCA TGA TCTCGACTCA CTGCAACCTC 788

Cys Arg Pro Gly Trp Ser He Met Ala * 510 515

CGCCTCCCGA GTTCAAGCGA TTCTCCTGCC TCAGCCTCCC GAGTAGCTGG GATTACAGGT 848 GCCCACCACC ATGTCAAGAT AATGTTTGTA TTTTCAGTAG AGATGGGGTT TGACCATGTT 908

GGCCAGGCTG GTCTCGAACT CCTGACCTCA GGTGATCCAC CCGCCTTAGC CTCCCAAAGT 968

GCTGGGATGA CAGGCGTGAG CCCCTGCGCC CGGCCTTTGT AACTTTATTT TTAATTTTTT 1028

TTTTTTTTTA AGAAAGACAG AGTCTTGCTC TGTCACCCAG GCTGGAGCAC ACTGGTGCGA 1088

TCATAGCTCA CTGCAGCCTC AAACTCCTGG GCTCAAGCAA TCCTCCCACC TCAGCCTCCT 1148 GAGTAGCTGG GACTACAGGC ACCCACCACC ACACCCAGCT AATTTTTTTG ATTTTTACTA 1208

GAGACGGGAT CTTGCTTTGC TGCTGAGGCT GGTCTTGAGC TCCTGAGCTC CAAAGATCCT 1268

CTCACCTCCA CCTCCCAAAG TGTTAGAATT ACAAGCATGA ACCACTGCCC GTGGTCTCCA 1328

AAAAAAGGAC TGTTACGTGG AAAAAA 1354

(2) INFORMATION FOR SEQ ID NO: 13:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 226 ammo acids

(B) TYPE: ammo ac d (D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Glu Glu Leu Thr Ala Phe Val Ser Lys Ser Phe Asp Gin Lys Ser 1 5 10 15

Lys Asp Gly Asn Gly Gly Gly Gly Gly Gly Gly Gly Lys Lys Asp Ser 20 25 30 He Thr Tyr Arg Glu Val Leu Glu Ser Gly Leu Ala Arg Ser Arg Glu 35 40 45

Leu Gly Thr Ser Asp Ser Ser Leu Gin Asp He Thr Glu Gly Gly Gly

50 55 60

His Cys Pro Val His Leu Phe Lys Asp His Val Asp Asn Asp Lys Glu

65 70 75 80

Lys Leu Lys Glu Phe Gly Thr Ala Arg Val Ala Glu Gly He Tyr Glu 85 90 95

Cys Lys Glu Lys Arg Glu Asp Val Lys Ser Glu Asp Glu Asp Gly Gin 100 105 110 Thr Lys Leu Lys Gin Arg Arg Ser Arg Thr Asn Phe Thr Leu Glu Gin 115 120 125

Leu Asn Glu Leu Glu Arg Leu Phe Asp Glu Thr His Tyr Pro Asp Ala

130 135 140

Phe Met Arg Glu Glu Leu Ser Gin Arg Leu Gly Leu Ser Glu Ala Arg

145 150 155 160

Val Gin Val Trp Phe Gin Asn Arg Arg Ala Lys Cys Arg Lys Gin Glu 165 170 175

Asn Gin Met His Lys Gly Val He Leu Gly Thr Ala Asn His Leu Asp 180 185 190

Ala Cys Arg Val Ala Pro Tyr Val Asn Met Gly Ala Leu Arg Met Pro 195 200 205

Phe Gin Gin Met Glu Phe Cys Ser Cys Arg Pro Gly Trp Ser He Met 210 215 220

Ala * 225

(2) INFORMATION FOR SEQ ID NO: 14:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32367 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: smgle

(D) TOPOLOGY: linear (n) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "COSMID: LLNOYC03 'M' 34F5"

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TTTCTCTGTC TCCATCCCTC TGTCTCTCCC TTTCTCTCTG TCTTTCCTTG TCTCTCTCTT 60 TCTCTCTCTC TCTCCATCTC TCTCTCTCCC TGTCTCTCTC TCTCCATCTC CCCGTCTCTC 120

CGTTTCTCTC TCTGCCTCTC CCTGTCTGTC TCTCTCTTTC TGTGTCTTAC ACACACCCCA 180

ACCCACCGTC ACTCATGTCC CCCCACTGCT GTGCCATCTC ACACAAGTTC ACAGCTCAGC 240

TGTCATCCTG GGTCCCCAGG CCCCGCCGGG GAGGAAGATG CGCCGTGGGG TTACGGGAGG 300

AAGGGGACTC CGGGCCTCCT GGTGCCCCAC TTTATTTGCA GAAGGTCCTT GGCAGGAACC 360 GTGACGCGTT TGGTTTCCAG GACTTGGAAA ACGAATTTCA GGTCGCGATG GCGAGCACCG 420

GCTTCCCCTG AAGCACATTC AATAGCGAGA GGCGGGAGGG AGCGAGCAGG AGCATCCCAC 480

CATGAAAACC AAAAACACAA GTATTTTTTT CACCCGGTAA ATACCCCAGA CGCCAGGGTG 540

ACAGCGCGGC GCTAAGGGAG GAGGCCTCGC GCCGGGGTCC GCCGGGATCT GGCGCGGGCG 600

GAAAGAATAT AGATCTTTAC GAACCGGATC TCCCGGGGAC CTGGGCTTCT TTCTGCGGGC 660 GCTGGAGACC CGGGAGGCGG CCCCGGGGAT CCTCGGCCTC CGCCGCCGCC GCCTCCCAAG 720

CGCCCGCGTC CCGGTTTGGG GACACCCGGC CCCTTCTTCT CACTTTCGGG GATTCTCCAG 780

CCGCGTTCCA TCTCACCAAC TCTCCATCCA AGGGCGCGCC GCCACCAACT TGGAGCTCAT 840

CTTCTCCCAA GATCGTGCGT CCCCGGGGCG CCCGGGTCCC CCCCCTCGCC ATCTCAACCC 900

CGGCGCGACC CGGGCGCTTC CTGGAAAGAT CCAGGCGCCG GGCTCTGCGC TCCTCCCGGG 960 AGCGAGGGCG GCCGGACGAC TGGGACCCTC CTCTCTCCAG CCGTGAACTC CTTGTCTCTC 1020

TGTCTCTCTC TGCAGGAAAA CTGGAGTTTG CTTTTCCTCC GGCCACGGAG AGAACGCGGG 1080

TAACCTGTGT GGGGGGCTCG GGCGCCTGCG CCCCCCTCCT GCGCGCGCGC TCTCCCTTCC 1140

AAAAATGGGA TCTTTCCCCC TTCGCACCAA GGTGTACGGA CGCCAAACAG TGATGAAATG 1200 AGAAGAAAGC CAATTGCCGG CCTGGGGGGT GGGGGAGACA CAGCGTCTCT GCGTGCGTCC 1260

GCCGCGGAGC CCGGAGACCA GTAATTGCAC CAGACAGGCA GCGCATGGGG GGCTGGGCGA 1320 GGTCGCCGCG TATAAATAGT GAGATTTCCA ATGGAAAGGC GTAAATAACA GCGCTGGTGA 1380

TCCACCCGCG CGCACGGGCC GTCCTCTCCG CGCGGGGAGA CGCGCGCATC CACCAGCCCC 1440

GGCTGCTCGC CAGCCCCGGC CCCAGCCATG GAAGAGCTCA CGGCTTTTGT ATCCAAGTCT 1500

TTTGACCAGA AAAGCAAGGA CGGTAACGGC GGAGGCGGAG GCGGCGGAGG TAAGAAGGAT 1560

TCCATTACGT ACCGGGAAGT TTTGGAGAGC GGACTGGCGC GCTCCCGGGA GCTGGGGACG 1620 TCGGATTCCA GCCTCCAGGA CATCACGGAG GGCGGCGGCC ACTGCCCGGT GCATTTGTTC 1680

AAGGACCACG TAGACAATGA CAAGGAGAAA CTGAAAGAAT TCGGCACCGC GAGAGTGGCA 1740

GAAGGTAAGT TCCTTTGCGC GCCGGCTCCA GGGGGGCCCT CCTGGGGTTC GGCGCCTCCT 1800

CGCCACGGAG TCGGCCCCGC GCGCCCCTCG CTGTGCACAT TTGCAGCTCC CGTCTCGCCA 1860

GGGTAAGGCC CGGGCCGTCA GGCTTTGCCT AAGAAAGGAA GGAAGGCAGG AGTGGACCCG 1920 ACCGGAGACG CGGGTGGTGG GTAGCGGGGT GCGGGGGGAC CCAGGGAGGG TCGCAGCGGG 1980

GGCCGCGCGC GTGGGCACCG ACACGGGAAG GTCCCGGGCT GGGGTGGATC CGGGTGGCTG 2040

TGCCTGAAGC CGTAGGGCCT GAGATGTCTT TTTCATTTTC TTTTTCTTTC CTTTCCTTTT 2100

TTTGTTTGTT TGTTTGTTTG TTTGAGACAG AGTCTCGCTC TGTCCCCCAG GCTGGAGTGC 2160

AGTGGTGCGA TCTCGGCTCA CTGCAACCTC CGCCTCCTGG GTTCAAGCGA TTCTCCTGCC 2220 TCAGCCTCCC CAGTAGCTGG GATTACAGGC ATGCACCACC ACGCCTGGCT AATTTTTGTG 2280

CTTTTAGTAA AGACGGGGAT TCACCATGTT GGCCAGGCTG GTCTCGAACT CCTGACCTCA 2340

GGTGATCCAC CCGCCTCGGC CTCCCAAAGT GCTGGGATGA CAGGCGTGAG GCACCGCGCC 2400

CGGCCTGGGT CCTGACGGCT TAGGATGTGT GTTTCTGTCT CTGCCTGTCT GCCTTGTATT 2460

TACGGTCACC CAGACGCACA GAGGAGCCGT CTCCACGCGC CTTCCCAGCG CTCAGCGCCT 2520 GCCGGGCCCC CGGAGATCAC GGGAAGACTC GAGGCTGCGT GGTAGGAGAC GGGAAGGCCC 2580

CGGGTCAGCT CGGTTCTGTT TCCTTTAAGG AACCCTTCAT TATTATTTCA TTGTTTTCCT 2640

TTGAACGTCG AGGCTTGATC TTGGCGAAAG CTGTTGGGTC CATAAAAACC ACTCCCGTGA 2700

GCGGAGGTGG CCGGGATCTG GATGGGGCGC GAGGGGCCCC GGGGAAGCTG GCGGCTTCGC 2760

GGGCGCGTCC TAAGTCAAGG TTGTCAGAGC GCAGCCGGTT GTGCGCGGCC CGGGGGAGCT 2820 CCCCTCTGGC CCTTCCTCCT GAGACCTCAG TGGTGGGTCG TCCCGTGGTG GAAATCGGGG 2880

AGTAAGAGGC TCAGAGAGAG GGGCTGGCCC CGGGGATCTC TGTGCACACA CGACAACTGG 2940

GCGGCATACA TCTTAAGAAT AAAATGGGCT GGCTGTGTCG GGGCACAGCT GGAGACGGCT 3000

ATGGACGCCT GTTATGTTTT CATTACAAAG ACGCAGAGAA TCTAGCCTCG GCTTTTGCTG 3060

ATTCGCAGAG TTGAGGTGCG AGGGTGAATG CCCCAAAGGT AATTCTTCCT AAGACTCTGG 3120 GGCTACCTGC TCTCCGGGGC CCTGCATTTG GGGTGTGGAG TGGCCCCGGG AAATAGCCCT 3180

TGTATTCGTA GGAGGCACCA GGCAGCTTCC CAAGGCCCTG ACTTTGTCGA AGCAGAAAGC 3240 TGTGGCTACG GTTTACAAAG CAGTCCCCGG TTTCTGACCG TCTAAGAGGC AGGAGCCCAG 3300

CCTGCCTTTG ACAGTGAGAG GAGTTCCTCC CTACACACTG CTGCGGGCAC CCGGCACTGT 3360

AATTCATACA CAGAGAGTTG GCCTTCCTGG ACGCAAGGCT GGGAGCCGCT TGAGGGCCTG 3420

CGTGTAATTT AAGAGGGTTC GCAGCGCCCG GCGGCCGCTT CTGTGGGGTT GCTTTTTGGT 3480 TGTCCTTCGC AGACACCGTT TTGCTCCTCT GAACTCTCTC TTCTCCCCCT GGCCGTGGAC 3540

CCGGGAGAGC AAAGTGTCCT CCAGACCTTT TGAAAGTGAG AGGAAAATAA AGACCAGGCC 3600

AAAGACCCAG GGCCACAGGA GAGGAGACAG AGAGTCCCCG TTACATTTTC CCCTTGGCTG 3660

GGTGCAGAAA GACCCCCGGG CCAGGACTGC CACCCAGGCT ACTATTTATT CATCAGATCC 3720

AAGTTAAATC GAGGTTGGAG GGCAGGGGAG AGTCTGAGGT TACCGTGGAA GCCTGGAGTT 3780 TTTGGGAACA GCGTGTCCCC GCCGAGCCTG GGAGCCCGTG GGTTCTGCAA AGCCTGCGGG 3840

TGTTTGAGGA CTTTGAAGAC CAGTTTGTCA GTTGGGCTCA ATTCCTGGGG TTCAGACTTA 3900

GAGAAATGAA GGAGGGAGAG CTGGGGTCGT CTCCAGGAAA CGATTCACTT GGGGGGAAGG 3960

AATGGAGTGT TCTTGCAGGC ACATGTCTGT TAGGAGGTGA AACAGAATGT GAAATCCACG 4020

TTGGAGTAAG CGTCCAGCGC TGAATGTAGC TCGGGGTGGG GTGGGAGGGC CCTGGTGTGG 4080 ATCGTGGAAG GAAGAAAGAC AGAACAGGGT GCTAGTATTT ACCCCGTTCC CTGTAGACAC 4140

CCTGGATTTG TCAGCTTTGC AAGCTTCTTG GTTGCAGCGG CCTTGCCTGT GCCCCTTTGA 4200

GACTGTTTCC AGACTAAACT TCCAAATGTC AGCCCCTTAC CCTTGACAGC AAGGGACATC 4260

TCATTAGGGC ATCGCGTGCT TCTCATCTGT GCTCAGCAGG CCCGAGATAG GAACAGAGGG 4320

GCGTTGGAGA TGCCACTTCC ACCAGCCCTG GGTTGAAGGG GAGCGAGGGA GACACCTTTT 4380 ACTTAAACCC CTGAGCTTGG TCAGAGAGGC TGAATGTCTA AAATGAGGAA GAAAAGGTTT 4440

TTCACCTGGA AACGCTTGAG GGCTGAGTCT TCTGCCCTTC TGACTCCCCC AGCAAATACA 4500

GACAGGTCAC CAACTACTGG AGATGAGAAA GTGCCATTTT TGGCACACTC TGGTGGGGTA 4560

GGTGCCCGAC CGCGTGTGAA AAAGTGGGAA GGAGAGATTT CTGCGCACGC GGTTCAGCCC 4620

CCAGGCGCGG TGGCGCATTC AGGTACTCAG ACGCGGTTCT GCTGTTCTGC TGAGAAACAG 4680 GCTTCGGGTA GGGGCTCCTA GCTCCGCCAG ATCGCGGAGG GACCCCCAGC CCTCCTGCGC 4740

TGCAGCGGTG GGGATAGCGT CTCTCCGTAG GCCTAGAATC TGCAACCCGC CCCGGGTCCT 4800

CCCCGTGTCC TTCCCGGGCG TCCCGCCGGG GATCCCACAG TTGGCAGCTC TTCCTCAAAT 4860

TCTTTCCCTT AAAAATAGGA TTTGACACCC CACTCTCCTT AAAAAAAAAA AATAAGAAAA 4920

AAAGGTTAGG TTATGTCAAC AGAGGTGAAG TGGATAATTG AGGAAACGAT TCTGAGATGA 4980 GGCCAAGAAA ACAACGCTCG TGCAAAGCCC AGGTTTTTGG GAAAGCAGCG AGTATCCTCC 5040

TCGGCTTTTG CGTTATGGAC CCCACGCAGT TTTTGCGTCA AAGCGCATTG GTTTTCGAGG 5100

GCCCCCTTTC CACCGCGGGA TGCACGAAGG GGTTCGCCAC GTTGCGCAAA ACCTCCCCGG 5160

CCTCAGCCCT GTGCCCTCCG CTCCCCACGC AGGGATTTAT GAATGCAAAG AGAAGCGCGA 5220 GGACGTGAAG TCGGAGGACG AGGACGGGCA GACCAAGCTG AAACAGAGGC GCAGCCGCAC 5280

CAACTTCACG CTGGAGCAGC TGAACGAGCT CGAGCGACTC TTCGACGAGA CCCATTACCC 5340 CGACGCCTTC ATGCGCGAGG AGCTCAGCCA GCGCCTGGGG CTCTCCGAGG CGCGCGTGCA 5400

GGTAGGAACC CGGGGGCGGG GGCGGGGGGC CCGGAGCCAT CGCCTGGTCC TCGGGAGCGC 5460

ACAGCACGCG TACAGCCACC TGCGCCCGGG CCGCCGCCGT CCCCTTCCCG GAGCGCGGGG 5520

AGGTTGGGTG AGGGACGGGC TGGGGTTCCT GGACTTTTGG AGACGCCTGA GGCCTGTAGG 5580

ATGGGTTCAT TGCGTTTGTT TTTCACCAAC AGCAAACAAA TATATATACA TATATATTAT 5640 ACAAATAACA AATAAATATA TATGTTATAC AGATGGGTAT ATTGTATATA TTATAGATAT 5700

TTGTTCGTCC TTGGTGCAAA GACACCCGGT GAACCCATAT ATTGGCTCCT GACTGCCTTC 5760

GGTTCCCCTG GGATTGGTTA TAGGGGCAAC ACATGCAAAC AAAACTTTCC CTGGATTATA 5820

CTTAGGAGAC GAAGCTACAG ATGCGTTTGA TCCAGAGTGT TTTACAAGAT TTTTCATTTA 5880

AAAAAAAATG TGTCTTTTGG CCCCTGATTC CCCTCCGTCT TCCCGTGTGG CTGCATTGAA 5940 AAGGTTTCCT TAGGATGAAA GGAGAGGGGT GTCCTCTGTC CCTAGGTGGA GAGAAACAGG 6000

GTCTTCTCTT TCCTCCGTTT TTTCACCTAC CGTTTCTATC TCCCTCCTCC CCTCTCCAGC 6060

CCTGTCCTCT GCTACAAACC ACCCCCTCCT CCCTCCGGCT GTGGGGAGCG CAGGAGCACG 6120

TTGGGCATCT GGATGAGCGG AGACTATTAG CGGGGCACGG GGGCTCCCCG AGGAGCGCGC 6180

GAATTCACGC TGCCCCATGA GACCAGGCAC CGGGGGGCGG AGGGGCCTTG GGTGTCCGCA 6240 GAGGGACGGG CGGGCAGAGC CTTCCTCCGC ATTCTAAACA TTCACTTAAA GGTATGAGTT 6300

TATTTCAGGG GTGCTGCTGG GAGAGCCTCC AAATGGCTTC TTCCAGCCCC TGCCTGACAG 6360

TTCAGCTCCC CTGGAAGGTC AACTCCTCTA GTCCTTTCTC CTGGTTCTGG GCAGGACAGA 6420

AGTGGGGGGA GGGAGAGAGA GAGAGAGAGA GAGAGAGACG GTCAGGATCC CCGGACCCTG 6480

GGGAACCCGT CAAAAATAAA TGAAATTAAG ATTGCCGACC AGAGAGAGAA CCGTGACAAA 6540 GCAAACGGCG TTCAAAGCAA AGAGACGAAC TGAAAGCCCG TTCCCGTAGG ACTGGTTATG 6600

AGGTCAACAC ATTCAAACAC AGCTTGCTCT GGATTTTGCT GAGCAGAGGA AGATACAGAT 6660

GCATTTGATC CAAAGTGTGT TACATCTTTC ATTATATGTG TGTCTATATA TATAAACATA 6720

TATAAATATA TAAACATACA TAAATGTATG TAAATATATA TAATCTATAT ACATATATAA 6780

ATATATAAAC ACATATATAA TATATAAATC TATAAACATA TATAATATAT AAACATAAAT 6840 ATATAAACAT ATATAATATA TAAATATATT AACATATATA AAATATGTAT AAATATATAT 6900

AAACATATAA ACATATATAA ATATATAAAC ATATAAATAT ATAAACATAT ATAAATATAT 6960

ACAAACATAT TGTATATATA TAAATATATA TAAAAACATA TATATACATA TAAAAATATA 7020

TATAAACATA TATACATATA AAGAAATATA TATAAACATA TATACATATA AATATACATA 7080

TATAAACATA TATATACATA AAATATATAT AAACATATAT ACATATAAAA ATATATATAT 7140 ATTAACATAT ATATACATAT AAAAATATAT ATATTAACAT ATATATACAT ATAAAAATAT 7200

ATATATATTT TTGGCCCCTG ATTCCCTTCG GTTCCTGTGG GATGGGTGAT TGAGTCAACA 7260 CATTCAAACA CAACTTTTCC ATCGATGTTG CTTAGGAGAT GAGGATACAG ATGCGTTTGA 7320

TGGAGAGGGT TTTACAAGCT CTTTCATTTA AATATATATA TATATATATA TATATTTTTT 7380

GGCTCCTGAT TCTCTTCCGT CTTCCCATGT GGCTGCATTT TAAAAGGCTT CCCTAAGATC 7440

GTTACGATTA AATCAACCCT CCCCAGGCAT CTTTACCGAG GGCTGTGGTC CCCAAAGCGA 7500 TACAGCCCAG GAGGGAGAGA GGCTTTGGTG ACTTGGAGGA AGGACTGTGT CCCTCCTTAG 7560

GGCGTCTGTG GCCTCAGTGA GGGAAGGAAG CTGCATCAGA CAGGGGTTTC CTCGCTGTCC 7620

ACCCCTCTGG CAGAAGATGG ATTGGGCTGC CCCGTATAAA TTAATGAAAA GATTAAAGTT 7680

TCGCTAAAGG GGACATCGAG TTTATGTGTC ATCTCCTGGT GTCTGTGTGC CTGGGATCTG 7740

CAATATATCC CAGCCCTTGA TGTACTGTTT CTATAAAAAT AAATTACTTG TAATTTAATT 7800 CCACACTATT TCTTTCCGTA GTCTATTACC GACGAGAGCA CGTTAGTTCA GCTGCGGAAA 7860

ATTGGTTGTG GGGTGTGTGC GGACCCCGAG AACGCCCTAA AATAAAGACA AATCGGGGAC 7920

AAGCTGGGGG TTATCGATTG CAGGGGTCGC ATGAAAATTT AACGACGGTA AATAATAATA 7980

AAAACAAACA TGGGAATGCA ATAAAAGACA TAATTCTCCA TCGCCGCGGG GGGAAAGGAT 8040

CCTATAGTAA AGGCGAGTGC GCTTTGAGGG GTCATAAAAA TCAATTAGTT CCAACACCCA 8100 CGTCCCGCGT TGAGGGGACG GGGACGAGCA GGGACAGAAA AAGAAACCAT ATTTGAATCC 8160

CATCTCTCTG TGAATTCTTG GGTCACATGC GTCTCAGTAC AGCCCGTCCC GTGCTGTGAC 8220

CGGATAGAGT TTCAATTTAC TGTGGAAATT TGCTGTAAAT AAATTGAGCA TCCGATAGAA 8280

GCTGTTGCTG ATTAACCTTT TATTTTTAGC GTGGCCCTGC AAAGTCGTAT CACCCAGCTG 8340

TCAGGCTTCT AATCGAAAGT TATGAGACCA CGGTGAGGGG CAGGCGGTAA TTTAATTACA 8400 ACAAATATCT TTGGGTTTAT GGCGCAGAGC TAAATTAAAT GTCATTATTC ACTGTCTGTA 8460

ATGGAAATCA AAAGGAAATC GCATTACGGC ATTTGGGAAA GAAAGCGGGG AGTGCTCTTT 8520

AATGAAGAAA TAACTGTCTT AAGCAGTGTC ACACACTTCA CTTACCATAT TCGGGCCTAA 8580

TTGGAATGGA TCGTGAATCA CTCCAAGACT GATTTATTAG CGCTTCACGC AGCGGCTAAT 8640

TCATCACTTG TATTCTTCAT CATTTTTTTT TTTCCTCTCG CCGTGTTGAA GGGAGAGTGA 8700 ATGAGGCTTT CCACGTTTCA GGAGGATTTT CTTTTTTGAA AAATGCCCTT CCAGAGGCTT 8760

TTGGGTGGCT GGCTTGCTTT CTGGGCCCTG GAGGAGACAG GCGGAGAGTC CAGGTGGGCA 8820

TGGAGAGGCA CAGTGGCAGG TCACCTGGAT GGTCAGTGGA GGTGGAGGTC TGAAGGCGCC 8880

AGCTTTGGAA ATTATTGGTG AATTTCGATG TCAGCACCAG GCAGGGGCCT TTTTGGCGGG 8940

GGTGTGAGGG AGGATGACTT TGCTGGGAAA CAGGATCAGG TTCTCCAGGC GCACTGCAGC 9000 CCGGTAGGAC CCACTTTGGA AATGAAAAGC CAGTTCCGAA AGCTGGGCTG GAAGCTTCCG 9060

TGTTGGGTTC AAGAGCAAGT TCACGTTGCG CTGTGTAGAC TCCTGGCTGC TCCCAAACTC 9120

TGAGGGTTTT CTGAGGTTCC CTTCATAGGG GCACCGGCCC TGGGCCATGC ACAGTGCGTA 9180

AGGGTGGCTG TGGGCCGAGG GACCCAGCAC GTGTTTTGCC CACAACAGCC GGAGTGACTG 9240 GTTCACTCAC CGCCTTGGCG GAGGACGCCT GTTCTCTGGA CGAATCATTT CTCTTGGGTG 9300

GTGACTGCCT TGTGGGTCAA GGTGCAGGTT TTCTGCCACA GAAAACCTGT TAGGAGGAAT 9360 TAAGCGACTA AGACTGTCAG GGAGGTGGTG GTGGGGGAGA GGAGGGGGTG GTGTCCAGAT 9420

TACCAGGCAT AGGCTAAACT GCCTGCACTC TCCAGCTGGT CTGTCTGTGG AGGAGGGGAT 9480

TGTCAATACT GGGAGAGCAG AGGAGGCTCG TAGGAGGTGA GAGGGGGTGG AATTTGCATG 9540

CAAATCTTCA CATGAGGCCT GTGTGAATTT CTCCAGCCTC CTGAGGGTCC CCTGCGCTAT 9600

TGCACTCAAC TTCTTGATAG TTTACCCCAA GACTCAGAAG TCCTTAGAGG GGCAGAATGC 9660 CCCCACCACA AAGCCTGCTA TCCTTGGGCG TCCTCAGGAC CCTTGGTCAT GAATGGGACC 9720

CTTTCATGTA TGGGGACCCT TGGTAATATG AATGGGACGC CTTCAGCTCC CCAGGGCTTC 9780

CGAGGAGGCC GAGAAGGGCA AAGACACTTC CGAGGAGGCC GAGAAGGGCA AAGACATTTT 9840

CTGGGCTTGG TGTGTCAAGA GCTAGATTGG AGAAGGGGCT GGATTTGGAA CTCTTTAGCC 9900

ATCAGCTCAC CCTCTCCGTT TGTGGCTAAA GTCTGAAGGT GGAAACTTCG GTTCTCCTAC 9960 AGGGTCTACA GGAGTTGGGG GGCGGGGCGC CCACACAGAA CGCTGGAAAG TTCGACAGTC 10020

CACTTCCACT GGCTCGGAAC TCACTTTTTC ACCTTAAGTT CATCAGCGGT AACGCATAGG 10080

TCTCACTTAG GCAGGGCACG GATGATTTAA CAATTTCTAC TTCTAGGTCA GGTGCGGTGG 10140

CTCACACCTC TAATCCCAGC ACTTTGGGAG GCCCAGGAGG GTGGATCGCT TGAGGTCAGG 10200

AGTTTGAGAC CAGCCTGGCC AACATGGTGA AACCCCGTCT CTACTAAAAT ACGAAAATTA 10260 GCCAGGCATG GTGGTGAGCA CCTGTAATTC CAGCTACTCG GGAGGCTGAG GCAGGAGAAT 10320

CGCTTGAACC TGGGAGGTGG ACGTTGCAGT GAGGTGAGAT CACACCACTG CACTCCAGCC 10380

TGGATGAGAG AGCAAGACTC TGTCTCAAAA ACAAAATAAA ACAAAAACAA AACAAAAATC 10440

AAAAAAGAAA ACCCAATTTC CAGTTCTAGG CCAGGTGCAG TGGCTCACGC CTGTCATCCC 10500

AGCACTTTGG GAGGCCCAGG AGGGTGGATC GCTTGAGGTC AGGAGTTCGA GACCAGCCTG 10560 GCCAACATGG TGAAACCCCA TCTCTACTAA AAATACAAAC GTTAGCTGGG TGTGGTGGTG 10620

TGCGCCTGTA ATCCCAGCTA CTCGGGAAGC TGAGGCTGGA GAATTGCTTG AATCTGGGAG 10680

GTGGAGGTTG CAGGGAGGCG AGATAGTGCC ACTGCAGTCC AGCCTGGACC AGAGAGCAAG 10740

ACTCCGTCTC AAAAACAAAA GAAAGCAAAA ACAAAAAACA AGAGACCAGC CTGGCCAACA 10800

TGGTGAAACC GCGTCTCTAC TAAAATACAA AATTAGCCGG GCATGGTGGT GGGCACCTGT 10860 AGTCCCAGCT ACTCGGGAGG CTGAGGCAGG AGAATGGCTT GAACCTGGGA GGTGGAGCTT 10920

GCAGTGAGCC GAGATAGTGC CACTGCACTC CAGCCTGGGC GACAGAGCGA GACTTGATTT 10980

CAGAACCACC ACCACCACAA CAAAACAAAA CAAAAAATCC AAAAAAACCC CAATTTCCAG 11040

TACTAGGTAG TCAGTGATGC AGGGCTGGAG ACAGAGGGGC GGTAAGTGTC TGGGCGCCCA 11100

CCATCAGTCA CCTCCCAGCT CCCAGAGGTG CAAAGTGCTT GGTTCAGCCT CATGGGAAGG 11160 ATGCTCCCTG GGGAGGCTGG GCTGGGTTCA CAGGGCTCTT CACATCTCTC TCTGCTTCTC 11220

CCCAAGGTTT GGTTCCAGAA CCGGAGAGCC AAGTGCCGCA AACAAGAGAA TCAGATGCAT 11280 AAAGGTGGGT GTCGGGACTG GGGGGACCTG AAGCTGGGGG ATCCTGCTCC AGGAGGGATG 11340

GGGTCGACGA GGTGCTGGCT ACACCCAGGA CCACCACACT GACACCTGCT CCCTTTGGAC 11400

ACAGGCGTCA TCTTGGGCAC AGCCAACCAC CTAGACGCCT GCCGAGTGGC ACCCTACGTC 11460

AACATGGGAG CCTTACGGAT GCCTTTCCAA CAGGTAGCTC ACTTTTTCTT CCTCTGAAGA 11520 TCCCTAGGGA CCTGCTGCTC CCTTCCCCTT TCCCCTATTT GCTGCCGCAT CCTGACACTC 11580

CTAGTCCCTC CCTGCCCCTG CAGACTTCTC AGCTGGCCCT TAGAAAAAAA GCCTCTTTTC 11640

CGAGGAGGCA TTTACAGGCA CCTTGGCACC TATGAAATCA GGCTGGGCCA GGCGGGGTGG 11700

CTCACACCTG TCATCCCAGC ACTTTGGGAG GCTGAGGAGG GTGCATCACC TGAGATCAGG 11760

AGTTCAAGAC CAGCCTGGCC AACTTAACGA AACCCCGTCT ATTAAAAATA CAAAATGGGT 11820 GTGGTGGCTC ACGCCTGTCA TCCCAGCACT TTGGGAGGCC GAGGCAGGTG GATCACCTGA 11880

GGTCAGGAAT TCGAGACCAG CCTGACCAAC ATGCTGAAAC CCCGTCTCTA CTGAAAACAC 11940

AAAGCTTAGC CGGGCGTGGT GGTGCACACC TGTGATCCCA GGTACTTGGG AGGGAGAATC 12000

ACTTGAACCT GGGAGGTGGA GGTTGCCGTG AGCCAATATC GCGCCACTGC ACTCCACTCT 12060

GGGTGACAGA GTGAGACTCC AAGACTCCAT CTCAAAAAAA AAAAAAAAAA TCAGGCTGTA 12120 AAAATCCACT TTTGGGAAGG TGAACACACA CAAGCCCAAA CAGAAATCTG ACAAAAACCA 12180

GAGGGGTGAA AAGTCCACAC AGTCAGGCAC CCCCACCTGG CTTGCTGCCT GGTTAAGAAG 12240

GGCGCAGATG CCTGTGCCTG GATACCAGAG ATGGGACAGA CACCCATTCC CTTTTCATCA 12300

CCACCCCCGA GTGCCCGAGG GCCTGGGGCG TCTGCCTGGC CCCTGGCCCC TGGCTTGGGC 12360

TCTGCACCTC TGAACTGGAG ACACCCTACT CAGCTCCCCA CTTACTTTGG AGTGAGCAGC 12420 GCTTGGGTGC CCAGCGTGGA TTTGGGGCTT CCAGGGAGTC GGGGTTCGGT CGCGGAGCCC 12480

AAGCTTCCCA AGGGCGCCCC CGCCCTGCCC TGGCTTAGTG GTGGGGATGG GATGGGGGGA 12540

AACGGGGAGC TGCGTGGAAG GAGGTGAAGG GTCACAGGAG GAGAGAGCGC AGCGCCCACG 12600

TGCGCCCTGC CTGAACGCGC AGCGCAGCGC CCGGCTGCGG TGCCCCTTGC CCCTTCGGTC 12660

CCTAATTTGG GGATCGGGAG TGCATGCGCG GGCGGAACGG GCTTGGGGGG GGGGCTCTGG 12720 CAGGGCGGAC GCGTGGCCTC CCTTCTTCAC CGTTTTATTC CAAGGGGACA GGCTGGGGAT 12780

TGTATTTGGG CGCGTGTTTG GCTGAGGGTG CAGGGACTTG GGGGGTGGCG GTGGGGAGCG 12840

CGGAAGGTAT AAACGTATAA ATCATAAGTA AACAACTCAG AAATGGACCC CGAGCGCTGG 12900

TCGCCGCTAG CTCTCCAGCT CTCCCTGGCC CAGGCCCGAA GGAGAGGGGT CCGCATCCCT 12960

CCGCGGTTCT CCTCTCCTGG GTACCTGGCC TTGAGGTGGG GGAACGAGCC TACTTCTTGT 13020 ACCGTCTTTT GCCGACGGCG GGACCCAGTG AAATTAGGCC GTTGGAGCCC GCAGGCCTGC 13080

CTGGCTTTGC GCACCGGAGT CTTGGGGACC TGGTGTCCCC GGGAAAAACT TGGGGACCTG 13140

GTATCCCCGG GAGAGGCTTG GGGACCTGGT GTCCCGGGAG AGGCTTGGGT ACCTGGTTTC 13200

TCTGGAAGAG GCTTGGACAC CTGGTGTCCT GGGAGGGCCT TTGGGACCTG GTGTCCTGGG 13260 AGAGGCTTGG AGATCTGTTG TCCTGGGAGA GGCTTGGGGA CCTGGTGTCC CTGGAGAGGC 13320

TTGGGGACCT GGTGACCTTG GAGAGGCTTG GAGACCTGGT GTTCTGGGAG AGGCTTGGGG 13380

ACCTGGTGTT CTGGGAGAGG CTTGGGGACC TGGTGTCTCT GGAAGAGGCT TGGACACCTG 13440

GTGACCCGGG AGGGCCTTGG GGATCTGGTG TCCCGGGAGA GCCTTGGGGA CCTGGTGTCC 13500

TGGGAGAGGC TTGGGGACCT GGTGACCTTG GAGAGGCTTG GGGACCTGGT GTCCTGAGAG 13560

AGCCTTGGGG ATCTGGTGTC CCAGGAGAGG CTTGGGGACC TGGTGTCTCT GGAAGAGGCT 13620

TGGACACCTG GTGTCCTGGG GAGAGGCTTG GGGACCTGGT GTCCTGGGAG AGGCTTGGGG 13680 ACCTGGTGTC CTGGGAGAGG CTTGGAGATC TGGTGAGCCG GGAGAGGCTT GGGGACCTGG 13740

TGTCCCGGGA GAGGCTTGGG GACTTGGTGT CCCGGGAGAG GCTTGAACAC CTGGTGTCCC 13800

AGGAGAGGCT TGGGGACCTG GTGACCTTGG AGAGGCCTGG GGACCTGGTG ACCCGGGAGA 13860

GCCTTGGGGA CCTGGTGTCC TGGGGAGAGC CTTGGGGACC TGGTGACCTT GGAGAGGCTT 13920

GGGGACCTGG TGTCTCGGGA GTGCCTTGGG GACCTAGTGA CCCGGGAGAG GCTTGGGGAC 13980 CTGGTGTCCC GGGAGAGGCT TGGGGACCTG GTGTCCTGGG AGAGCCTTGG GGATCTGGTG 14040

TCCTGGGGAG AGGCTGGGGG ACCTGGTGTC TCGGGAGAGA GCCTTGGGGA CCTGGTGACC 14100

CGGGAGAGGC TTGGACACCT GGTGTCCCGG GAGAGGCTTG GGGACCTGGT GACCCGGGAG 14160

AGCCTTGGGG ACCTGGTGTC CTGGGGAGAG GCTGGGGGAC CTGGTGTCTC GGGAGAGAGC 14220

CTTGGGGACC TGGTGACCCG GGAGAGGCTT GGACACCTGG TGTCCCGGGA GAGGCTTGGG 14280 AGCCTGGTGT CCCGGGAGAG CCTTGGGGAC CAGGTGACCT TGGAGAGGCT TGGGGACCTG 14340

GTGATCTTGG AGAGGCTTGG GGACCTGGTG TCTCGGGAGA GGTTACGGGG GCTGGTTGGG 14400

GGAGAGAACG TTGTGAGCCA AAGTCCCTGA ATCCCTGCGA AAAGAGCGCA TCGGGAGCTC 14460

CCCCTGAGGG CGTTCCATTT GTGGACCCCC CTCCCATGCG CTTTGCAGGG AGCTGTTCGG 14520

ATTCCCCTGG CCCGGCTCCC GCGGATGCAT CCAGTGGCAG CGCCAATTCT GGGCCAGGGG 14580 GAAGGAGGAA AGGCGGGTGT GGGGTGGTCT CCACGGCTGG AGAAGGGGCG ACGCTCCCTA 14640

GGGGAGAAGA GGCACGTTGG GGGTTTCCGG GGGCGCGGGG CGGAGCAGGC CCCCCAGTCC 14700

CCATCCTGCG CCCTCACCCC GCCGGGTCCG CTCCCGCAGG TCCAGGCTCA GCTGCAGCTG 14760

GAAGGCGTGG CCCACGCGCA CCCGCACCTG CACCCGCACC TGGCGGCGCA CGCGCCCTAC 14820

CTGATGTTCC CCCCGCCGCC CTTCGGGCTG CCCATCGCGT CGCTGGCCGA GTCCGCCTCG 14880 GCCGCCGCCG TGGTCGCCGC CGCCGCCAAA AGCAACAGCA AGAATTCCAG CATCGCCGAC 14940

CTGCGGCTCA AGGCGCGGAA GCACGCGGAG GCCCTGGGGC TCTGACCCGC CGCGCAGCCC 15000

CCCGCGCGCC CGGACTCCCG GGCTCCGCGC ACCCCGCCTG CACCGCGCGT CCTGCACTCA 15060

ACCCCGCCTG GAGCTCCTTC CGCGGCCACC GTGCTCCGGG CACCCCGGGA GCTCCTGCAA 15120

GAGGCCTGAG GAGGGAGGCT CCCGGGACCG TCCACGCACG ACCCAGCCAG ACCCTCGCGG 15180 AGATGGTGCA GAAGGCGGAG CGGGTGAGCG GCCGTGCGTC CAGCCCGGGC CTCTCCAAGG 15240

CTGCCCGTGC GTCCTGGGAC CCTGGAGAAG GGTAAACCCC CGCCTGGCTG CGTCTTCCTC 15300 TGCTATACCC TATGCATGCG GTTAACTACA CACGTTTGGA AGATCCTTAG AGTCTATTGA 15360

AACTGCAAAG ATCCCGGAGC TGGTCTCCGA TGAAAATGCC ATTTCTTCGT TGCCAACGAT 15420

TTTCTTTACT ACCATGCTCC TTCCTTCATC CCGAGAGGCT GCGGAACGGG TGTGGATTTG 15480

AATGTGGACT TCGGAATCCC AGGAGGCAGG GGCCGGGCTC TCCTCCACCG CTCCCCCGGA 15540 GCCTCCCAGG CAGCAATAAG GAAATAGTTC TCTGGCTGAG GCTGAGGACG TGAACCGCGG 15600

GCTTTGGAAA GGGAGGGGAG GGAGACCCGA ACCTCCCACG TTGGGACTCC CACGTTCCGG 15660

GGACCTGAAT GAGGACCGAC TTTATAACTT TTCCAGTGTT TGATTCCCAA ATTGGGTCTG 15720

GTTTTGTTTT GGATTGGTAT TTTTTTTTTT TTTTTTTTTT GCTGTGTTAC AGGATTCAGA 15780

CGCAAAAGAC TTGCATAAGA GACGGACGCG TGGTTGCAAG GTGTCATACT GATATGCAGC 15840 ATTAACTTTA CTGACATGGA GTGAAGTGCA ATATTATAAA TATTATAGAT TAAAAAAAAA 15900

ATAGCCGTGC ACTCTTGACC CCGTCAACGT CCAACGTGGA AAAGGCGTTA CCTCTTCTCC 15960

CAGCGCTGGC CGCCTGGCCA CTGAGGGCCC TTTGCAAAAA TCACGGGTGT AGAGATGGCC 16020

CTGGGCGCGC TGGGAGTGTG GTTGTGTTTC TGAAGGGGAT AAAAGAGGGC ACGGTGGTGC 16080

CAAGATATCA GTTTGGTACC TGAGCTGTTT CTGGTTGGGA AGCGTAAAAG CCAGGGAGAG 16140 ATCCAGAGAG TTTTCAAGTT TTTGCAGATG TAGGTGGTTC CAGCTTTTCT TTCTCCCCTA 16200

CTCCATCTTC TGCGTTCCCC CAGTTCTTTT ATTTCTTTGT TTTTTATTTT TGAGACAGAG 16260

ACTTGCTTTG TCGCCCAGGC TGGAGTGCAG TGGCGCAATG TCAGCTCACT GCCACCTCCA 16320

CCTCCCGGGT TCAAGCGATG CTCCTGCCTC AGCCTCCCGA GTAGCTGGGA CTACAGGCAC 16380

CTGCCACCAC CCCCGGCTAA TTTTTTGTAT TTATAGTAGA GACGGGGTTT CACCGTGTTG 16440 GCCAGGCTCG TCTCGAACTC CTGACCTCAG GTGATCTGCC CGCCTCGGCC TCCCAACGTG 16500

CCCCCAGTTT TATAAACAGC AGATAGCAAC TTGTCGTCAC AGCTGGCATG GGCTGGACAG 16560

TTGCTTGAAA TGACCTAACC AAAAACATTC AAGGGTTCTG CCCCCAGATT TCGGGAGATC 16620

CACGTTCCAT GTTCTGATTG GTTTTCTGGG AACACAGCAA GGGGTTTGGT GACCTCCGAG 16680

AAGATCCATC TGCATGATTG GCATTAGTTA CCACAGCCTG CCCAGAGAGA AACTATCTTC 16740 TCCCAACATT TACTAACATC CACTGGTCAA CTCTCTTATT TCCATAACAC ATTTGCATCT 16800

TTCTGGATTC AAGCTTGGTG GTTTTCTTTC CTAACTTCTG ATTTAGATAC TTCTCCCTGA 16860

GGTGGGGATA AAAGAAAAAA AAAAAACAAC TTCTTTTTTT CTTCCGCATA ACACTTTCTA 16920

TCTTGTCACT GAGCTGAACT GTAGATCCAT TTGGACCCGT CTCATTTGTA TCTTCTGATA 16980

TTCTTTATAC AAACCAAAAG TCCCCTTCAA CATTTTTTAT GTCAAAATGT TACAACCGCT 17040 GTAAAATGAC GGAGAGAGAG AGAAAGAATC CCAGACATTA ACGGTATTAG AGAGTTTGCC 17100

TCATTCATCC ATTTTTCTTA AAAGCTGGAA ATTAAAAAAA AAAAAGAGAG AGAGAGGCTT 17160

TAATAGTTAA GCTGAAATTT TTATCGAAAA GAAGAATTGC ATTTTGAATC TTTGGGAAGT 17220

AGGTTCATTC ATCAGAGTAT GTAACCCTTT GGAAAAGTGG TTGGTAAGAT ATGTACAGCC 17280 CTAGATTTTT TTTTTTTTAA CCAAAAAGGC TGAGTAATTT TGAAAAATCG AAACATAACA 17340

GTGTGTCATC ATTTCCTCCC AAGAAAAAGC TCACTCCACG TGAGTAGAAA GACATCTACC 17400 TGGTCCCTGT AGAATCTGAA CGTTTCTCTT TAGAGACGGA ATTTCAATCT TGTTGCCCAG 17460

GCTGGAGTGC AGTGGCACAA TCTCGGCTCA CCGCAACCTC CGCCTCCCGG GTTCAAGCCA 17520

TTCTCCTGCC TCAGTCTCCC GAGTAGCTGG GATTACAGGC ACCTGCCACC AGGCCTGGGT 17580

AACTTTCTGG TATTTTTAGT AGAGACAGGG TTTCAGCCTC CCGAGTAGCT GGGATTACAG 17640

GCACCTGCCA CCAGGCCTGG GTAACTTTCT GGTATTTTTA GTAGAGACAG GGTTTCAGCC 17700 TCCCGAGTAG CTGGGATTAC AGGCACCTGC CACCAGGCCT GGGTAACTTT CTGGTAGTTT 17760

TAGTAGAGAC AGGGTTTCGG CCTCCCGAGT AGCTGGGATT ACAGGCACCT GCCACCAGGC 17820

CTGGGTAACT TTCTGGTATT TTTAGTAGAG ACAGGGTTTC GGCCTCCCGA GTAGCTGGGA 17880

TTACAGGCAC CTGCCACCAG GCCTGGGTAA CTTTCTGGTA TTTTTAGTAC AGACAGGGTT 17940

TCGGCCTCCT GAGTAGCTGG GATTACAGGC ACCTGCCACC AGGCCTGGGT AACTTTCTGG 18000 TAGTTTTAGT AGAGACAGGG TTTCAGCCTC CCGAGTAGCT GGGATTACAG GCACCTGCCA 18060

CCAGGCCTGG GTAATTTTTT TGCATTTTTG GTAGAGACAG GTTTTTGCCG TGTTGGCCCG 18120

GCTGGTCTCA AACTCCTGAC CTCAGGTTGA CCTGCCCGCT TTGTCCCTCG CAAAGTGCTG 18180

GGATTACAGG CGTGAGCCAC CACACCTGGC CTGAATCTGA ACTTTTAAAA GGGAGTTACT 18240

GACTCTCAAC TGTGCGGGGA CGGTTTCACT TTGATTTAAT ATGGAAAGAG GGCCAAGTGT 18300 CATCCTCACA AATGGGTCCC CGAAGCAGAT CAAACGCAGA GAACTGTGAG GGTGGGACAC 18360

GAGTGTCTGT GGACACTGGC TGCCTTTGGC TTTTCTCCTG CGAGAGAAGT TGGGTGACTT 18420

TCTGTAGGTG GATGAGTGAT CCCTGAATGA GTGTGGGGTA CGTGTATGCT AGCTGCTTCT 18480

TTCTCCCTGA AACTCTCGGA TGGAAGGAAG TAAGAAATTC AGCTTGGGCT GTGACCAGTT 18540

CTCACCACCA ACGCCCTCTT CTCTCTCCCT TCTCCTTCCT TCCTTCCTTC CTTCCTTTCT 18600 TTCTTTTTCT TTCTTTCTCT CTTTCTTTCT TTTCTTTCTT TCTGTTTCTT TCCTTTTTAT 18660

CTTTCTCTCT TTTTCTTTCT CTTTTCCTTT TTTGTTTCTT TCTTTCTTTT TCTTTCTTTC 18720

TTTTTCTTTC TTCTTTCTTT CTTCGATGAA GTCTCACTCT GTCACCCAGG CTGGAGTGCA 18780

GTGGTGCAAT CCCAGCTCAC TGCATCCTCT ACCTCCTGGC TTCAAGAAAT TCTCCTGCCT 18840

CAGCCTCCCA AGTAGCTGGG ATGACAGGCA CCCACCACCA TTCCCGGATA ATTTTTGTAT 18900 TTTTTAGTAG AGACTGGGTT TCGCCATGTT GGCCAGGCTG GTCTTGAACT CCTGACCTCA 18960

CATGATCCAC CCGCCTCAGC CTCCCAGAGT GCTGGGATTA CGGGGTGAGG CACCGCGCCC 19020

GGCCTCCTCT CTCTTTTTCT GAGATGTTTA GGAAGGACTG GGCTGATGGG GACCCTCTGT 19080

ATGTGATGTG CGTGGGTTTG GTTTCCCGGA AGGCCCTCCA GAGACACGTT TGCGTGAACA 19140

TTCAGCATGG AAACAACATA CGTCTCTCCA CAGGAGGTGA GAAATTGAAT TTATGGGGTG 19200 GGTGTACGCT GGCGATTCTT GGTGCTTTTT GCTCAAAACA AGGTTCTTTT GAAAGTCACG 19260

TTCCTGCTTT CCCTGTGGCT TCCCGGTGAG CTCGCTCGCA GAGCAAGGAA TACCACCCAG 19320 AGAGCAACGT GGGCTGTGTT CCGTTGTAAC GCCGTTGCAG AGAGAGGATT TGGTGTGTGA 19380

GATCCGTACC AGCTCCAGCA CACTGATAGG AACACGTTGC TGGCCGAACT GAACGATGCT 19440

GGGTTGGGTC CTGATTGATA CGTATTTTCT TCCCTCCTCT CCCCAAAACT TGGCCAAATA 19500

GTCCGTGGAG GGTTGTCAGT CGCCGCAGTT GAGCAAAAAA CACTTCTTCC TTTGAGTGGC 19560 TGTTCTGGTG AAATCTGTTT CTGACATATC CACTTTTCTC TCTCTTTTCT CTCTCTCTGA 19620

CTGCGAAGCA CCCACAGGGA GAAGGAATTG GATGTATCGG ATGTTGGTAT TAGATTTTCT 19680

TTCTCCGTTC GAGTCTCTGA CTGGTGCATA CTTTGCAAAG GTGTGTTCCT GGCAATTGCC 19740

AAGAGTTAGA AAAATGCACC TTCTCTGGTG GCCGTTGGGG TGTTGTTTCA CAGGCAGTGG 19800

TGACAGGGCC CCTTGGCTGT GGCTGTCTTC TCCAGCGCCG TGGATAAAGA GACGGGACAG 19860 ATTCTGTGCC TCTGTACGAT TTAGAGCGTA ACTGACCGCG TCCAACACCC GTTTTTCCAC 19920

TTACAAAGCT GGTGGTGCGA CGGGCTTGGT GTCTCCCGTA CGGGAAGGAG GCCTTTGGGC 19980

CGCTCCAAAG ACGCCCTGTC GTAGGAATGG CCTCTCCATC CCGCCAAAGT CCAGCCAGGC 20040

CCCCGAAATG GTCCCATTTC CTTGGAAGCC TGAGTTTCTG TTCTGGTCTT GCTGCTGTCC 20100

TTGGCCACGT CAGCACGTGG GAGCATCTGT GGATACCGCA GAGTCTGGGG ACAGCTGGGC 20160 GTTTAACCGA AATGAAGCCG AGACGGGTTT CAGGTTTTGG TGCCAAGCTC TGGTCAGGAT 20220

GAAAGGGAAA TACCAGAGTC CTCTGTCCTC GCCTCTGGGT TTCATGCTGA CCTTTCTAAC 20280

ATTTGTTTTC CCCTAAGAAC AAGCAGAAGC CTCCAGCTCC CTTTAGCTCC ACAGTTTTCC 20340

CGGGGACATA GCGAGGATGG CACACGGCAG CCACTCCCAC GACACACATT TCGGAGGCAC 20400

TTTGCTGGAA GCCGCTTGTC TCCTCCAGCT TTGGGAGGTC TGGGGAGGAG AGAGGCTTTC 20460 GGTGGACACG TTTGACATTA AAAAAAAAAA AAAAAAAAAA AAAAAAACTG GTGCCTAATT 20520

TATTAAAGAG AATTAGCTTA GCGAGTATAT GCTGATATTC TTCGACACAC GTGGGTAAGT 20580

TGATGCCATT TATAAATGTT TTATTGAAAT TTGATATTTA ATGAGAAGCC GGTTAAGGAA 20640

TGTAGACAAT ATCCCGTTTC AAAGCTATGA AATGTGCTAT TTATTGAAAG GGGATGTGGC 20700

TTCACGAGTT CAGCCCATTG TACGTGCAGG TCCCGTGGGA AGGAGGCAAA AGCCCCTGCT 20760 TCTTACTTTG TGATGTATGT GCATTTGTTA TTTATTTTTT TTTCCTTGGT CGGACGTTCA 20820

TAAATATGTA CTATTTTAAT TATGTCGAGT GTAAATTTGA CATCGCGTTG CATTTATTTT 20880

TATATTTCTG AAAACTGTTG CTTTTTCTTT TTCCCTCCCC CATTGACGAC ATAGCGGCCC 20940

CCGCGTCCGG GTTACAAATA CATCTACAGA TATTTTCAGG GATTGCTTCA GATGAAAACA 21000

AATCACACAC CGTTTCCCAA ACCAACAGTC TTCACATTTC TATCCCTCTG TTATTGTCGG 21060 CAGGCGGTGA GGGGTAGAAA AAAAACAAAC AAACAAACAG AAAAAAAAAC CAAAAAAAAC 21120

CACCCTGAGT TTCTCTGGTG ACGCCCTCAT TCTCCTAACG TTCAATAATC TCAATGTTGA 21180

GTTGCAGCAA CAGACTGTAT TTTTGTGACG CCCCGTAGTA TGAATGTACA TCTTGTAAAA 21240

CTGAGATATA AATAAACTTA TAAATATTTG TATTCAAGTG TTAAAAAAAA AAAAATTCTC 21300 AACCTCTCCC CTGAGGACAG GCTTATTGGA AAAAAAAAAA AAAAAAAAAA ATCCTGAGTC 21360

GGCCGTGGCT GAACACAGAG TGTTGTTCTG CTCCGTGCAT TTCCAGGGTG GGTACCCAGT 21420 GTTGCCCCCC AGCCTTAGAT CGGGAGGTAC CATTGACTTT TGCTTGTATC CCATCCCCTT 21480

CCTTTACTGA AACCTACCTC CCCGCTTCTC AGCCAACGTC CCCCCAGAAG GTGGCAAAAA 21540

AAACAGAGGA AAAAGCCCTG ATTTGAATCA AGTCAGAGCT GCTAATTCTC CACTTTCTTT 21600

AATTAATTAA TTTATTTTTT TTTTTGAGAC TGAGTCTCGC TCTGTCGCCC AGGCCGGAGG 21660

AGTGCAGGGG CGCGATCTCG GCTCACCGCG ACCTCCGCCT CCCGGGTTCA AGCGACTCTC 21720 CTGCCTCAGC CTCCCGAGTA GCTGGGATGA CAGTCACCTG CACCACCGCG CCCGGCTCAT 21780

TTTTGTATTT TTAGTAGCAA TGGGGTTTCA CCGTGTTGGT CAGGCTGGTC TCGAACTCCT 21840

GACCTCGTGA TCCACCCGCG TCTGGGCCCG GCCGGTGATG TGTGTGCTTT TAACTTTTAT 21900

TTTGTTCCAG TTTTCGACAG TGGCACGGAT TTTCCAGCAC GGTCTTGCAA GGATGATTGA 21960

GTCATTTTTG AGACAAAAAA TATAATAATA ATAAATGGAA AAAGAAATCG ACTTTTAAAA 22020 ATGACAAATT TTTTTTTTTT TTTTTTGCAT AGATTTTTCT CTCTTTATGT AAAGGAAAGT 22080

TCATGATTGG ATTTGGCCGG CCTGACTGCT TCCCGGCTGT GATAAAAAAC ACATGTGAGC 22140

TGGGAGGGAA GTGGGGGAGG GACACAGCTG CCCACACAGG GTTCCCACCG CGGTTACAGG 22200

GTGGGCAGTG CTGGGGGAGC TTTCTCTGTG GGGGGCTCAG AGCCTGAGGA CAGGTGAGCC 22260

TCTCCGACAC CTCCCCAGTT GCCTGGAGTC TAAACCGTCC GTTGTCTGTA CCGTCCGTTC 22320 TTCCTGCTGA CTCCTGGTAG TTCCTGAAAG CTTCTCTTGG CCAGAGAAGG GGTTTCAGAG 22380

GCCGTGTGTC CAGGCCATTC TGCAAAGTGC AACTTGACCG TTCCTTTCCT TTTCTGGCCT 22440

GCGTGGTCTG AAGCTCAGAG CCCTCTCTTC ACCCAGCCTG TGTGTGTCTT GCCGGACAGA 22500

AGAAAAATGG TGCTTTTTGC GTGTTAGCAG AGGTGCTTTT CATGGCTGAC CTCAACGCGT 22560

CCATCTCCAG CCTTGACCAA GCTGTTTTTT AGGGGCAAAC GCAGGCAAGT TCTGAATGCA 22620 CACAGTTATT TCATGGTTAA ACTATTCAGC TTTGGCCGGG CGCAGTGTGG CTCTCACGCC 22680

TGTCATCCCA GCACTTTGGG AGGCCGAGGC GGGTGGATCA CCTGAGGTCA GGAGTTCGAG 22740

ACCAGCCTGG CCAACACGGT GAAACTCTAT CTCTACTAAA AATACAAAAA TTAGCCGGGC 22800

GTGGTGGTGT GTATCTGTAA TCCCAGCTAC TCAGGAAGCT GAGGCAGGAG AATCGCTTGG 22860

ACCCAGGAGG CGGAGGTTGC ACTGAGCCGA GATCGCGCCA TTGCACTCCA GCCTGGGCGA 22920 CAGAGCCAGA CGCTGTCTCA AAAAAATGAA TAATAAAATA AAATAACAGG AACTAAATAA 22980

AATAAAACGT TCAGCTTTGT TCTGCAAATC CACTCCTATT GTTTTACGTG GTTTGAGAGA 23040

CTCTGTCCCT TAGAAATAGA TGTTTGTTGC CAATTGTAAT GAATCTGTTT CAAAAATGAA 23100

CAGAATATTC AAATGGTTTG AGAGATCTTT TCCCTTAGAA ATAGCTTGTT GCCAATCACA 23160

AAGAATGTTT TTCAAAAATG AATGGAATCT TCCTGGATAT CGCTTCCAGA TCTTCATTTT 23220 TTTTGCATAG TTCAACCTGA AAAGTAAGTG TCTCAGCCCT GAATTTCTTT CTGATTTTTC 23280

CATGGGTTGT CTTGCAGACT TCTCTGGACT TGACCACATT TAAAAAAAAA AAAATTAACT 23340 TTTTCACACG GACACGGTTT CAATAGGAAT GAGATCTTTG AGTTTTTATG TAACAGATTC 23400

TTACCATCAG TTCTCAGATT CCCAAATTAC ACACAAAAAG CCACGGACTT CGCCTCCTGC 23460

TAACATGTCC TTCTGTTTCT GAGGCTTCTG TTGGTGTTAG ACTTTCATGT TTAATAGCAG 23520

ACAATGTAGG GATTTAAAGA AAAATGCAGA GAAAGCAAAA ACACTGACCA AACACACGGA 23580 GATAAGCTTT CTAAAGCCTT TGTTCTTGGA GTTGTCGTTA AAAAAAAAAA GTTGTTTTAA 23640

ACTTTGCAAG CATGCCTATA TTGAACTCAT AAGCAAGAGA GCCAAGAAAA ATAGTGTCGG 23700

TCGTCTACTC TACACGTTTT CCCAAAACAG ACGTATTTTA ATTTCTTTTG TTTGAACTCA 23760

CAGATGCTGA GAGTTAAAAG TTAAATTTTT GTCATGAACA ATAGTGGCCA AAACCACAGT 23820

TACTTTTGCA CTATAGCATA ATAAGAAAAA TACAGGCTGG GCTCGGTGGC TCACACCTGT 23880 AATCAAAGCA CTTTTGGAGG CGAAACAGCC AGATCCCTTG AGCCCAGGAG ATTGAGACCA 23940

GCCTGGGCAA CATAGCGAGA CCCTCATCTC TACAAAAAAG GTTTGTTACA TATGTAACAA 24000

ACCTGCACAT TGTGCACATG TACCCTAAAA CTTAAAGTAT AATAATAAAA AAATTAAAAA 24060

AAAATTCACC AATCAACTGC CTGCTGGTGC CTTCAAGAGA CTCACCTAAC ACATAAGGAC 24120

TTGCATAAAC TTATAAAACA ATTCAATGGA AGAATCCTTG AAAGTATTCT GAGAAGACAG 24180 TATAATAAAC TGATTTCTAA AAAGGCTATA AAAAATTGAA TAAATCATTG TTGGGCATCC 24240

TGTGCTGAAA TATAATGCAG CCAATAAAAA TTACAAAATG AATAAACATT TTATAACAAT 24300

AAAAAAAAGT CAAATAATTA GGCAGGCATG GTGGTGCTCT CCTACGGTTG AAGCTATTCA 24360

GCAGGCAAGA GGATACTTTG TTTTTGTTTT TTAATTTTTT TTGAGACAGA GTCTCGCTCT 24420

GTTGCCAGGC TGGAGTGCAG TGGCGTGATC TCAGCTCACT GTAATTTCTG CCTCCCGGGT 24480 TCAAGCGATT TTCCTGCCCC AGCCTCCCGA GTAGCTGGGA TTACAGGTGC CCGCCACCAC 24540

ACCTGGCTAA TTTCTTTTGT ATTTTTAGTA GAGACGAGGT TTCCCCATGT TGGCCAGGCT 24600

GGTTTTGAGC TCCCGACCTC GGGTGATCCA CCCGCCTCAG CCTCCCAAAG TGCTGGGATG 24660

ACAGGCGTGA GCCACCGCGC CTGGCCCAGG AGGATTATTT GATCCCAGGA GGTGGAGGCT 24720

GCAGGAAGCC ATGATTGCAC CACTGCACTC CAGCCTGGCT GACAGAGTGA GACCACATCT 24780 CTAAATAAAT GAATAAATAC AGGCAGAAAC TTTTTTTGTT TTGTTTTGAT GGAGTCTTGC 24840

TCTGTCACCA GGCAGGAGTG CAGTGGTGCC ATCTCAGCTC ACTGCAACCT CCACCTCCTG 24900

GGTTCAAGCA ATCCTCCTGC CTCAGCCTCC CGAGTAGCTG GGATTACAGG TGCCCGCCAC 24960

CACGCCCGGC TAATTTTTTG TATGTTTAGT AGAGACGGGA TTTCACCGTG TTAGCCAGGA 25020

TGGTCTTGAT CTCTTGACTT TGTGATCTGC CTGCCTCAGC CTCCCAAAGT GCTGGGATTA 25080 CAGGCATGAG CCCAGGAGTT CAAGACCAGC CTCAGCAACA AAGTGAGACC TTTTCTCTCC 25140

AAAAAATCAA AAATTTAGCC AGCTGTGGTG GCTCCTGCCC GTGATCCCAG TACTGTGGGA 25200

GGCTGAGGCA GAATTGCTTG AGCCCAGGAG TTCGAGACCA ACCTCAGCAA AAAGGACTCT 25260

CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTATAT ATATATATAT ATATATATAT 25320 GAGTTTCAAA AATTGCTGGG TGACCAGCTC ATCTACTGGT TTTCCCCTTG GGAAAGTGAA 25380

ATTGTCATGT ATTGAAGATT TCCAAGGAAG TTGTATTGAA TGAGAAACAA ACTCAATCTG 25440

TTCGTGTTTA AAGAGCTGCA GTGCGTTTGC TGTGTTTCCC ATAAAACTGC ACTTCCAAAA 25500

GACACGCTGA GAAAGGAGAC CAGGATTTGT AATTCAGAAA TTGGAAAGCA AGTTAGGCTG 25560

GACGTGGTAG CTCATGCTTG TTGTAATCTC AGCACTCTGG GAGGCTGAGG CAGGAGGATC 25620

ACTTGAGCCC AGGAGTTCAA GACCAGCCCG TGCCACATGG TGAAACCCTG TCTCTCCAAA 25680

AAATAAAACA TTTAGCCAGA TGTGGTGACT CATGCCTGTA ATCCCGGTAT TCTGGGAGGC 25740 TGAGGCAGAG TTGCTTGAGC CCAGGAGTTC AAGACCAGCC TCGGCAACAA AGTGAGACCC 25800

TGTCTCTCCA AAAAATAAAA CATTTAGCCA GCTGTGGTGA CTCATGCCTG TAATCTCAGT 25860

ACTCTGGGAG GCTGGGGCAG AATGGCTTGA GCCCAGGAGT TCGAGACCAA CCTCAGCAAC 25920

AAAGTGAGAT CTTGTTTCTC CAAAAAATCA AAAATTTAGC CAGCTGTGCT GGCTCATGCC 25980

TGTAATCCCG GTACTCTGGG AGGCTGAGGC AGAATCGTTT GAGCCCAGGA GTTCGAGACC 26040 AACCTCAGCA ACAAAGTGAG ATCTTGTTTC TCCAAAAAAA TCAAAAATTT AGCCAGCTGT 26100

GCTGGCTGGT GCCTGTAATC CCGGTACTCT GGGAGGCTGA GGCGGAATTG CTTGAGCCCA 26160

GGAGTTCAAG ACCAGCCTCA GCAACAAAGT GAGATCTTGT TTCTCCAAAA AATAAAACAT 26220

TTAGTCAGCT GTGGTGGCTC AAGCCTGTGA TCCCAGCATT TTGGGAGGCC GAGGCGGGCG 26280

GATCACGAGG TCATGAGATC GAGACCATCC TGGCTAACAC GGTGAAACCC CGTCTCTACT 26340 AAAAATACAA AGAAAATTAG CCGGGCGTGG TGGCGGGCGC CTGTAGTCCC AGCTACTCAG 26400

GAGGCTGAGG CAGGAGAATG CCGTGAGCCT GGGAGGCGGA CCATGCAGTG AGTCAAGATC 26460

GCGCCACTGC CCTCCAGCCT GGGCCACAGA GCAAGACTCC GTCTCAAAAA AAAAAAAAAA 26520

AAAACTGCTG CCCAACCTGT GTTTGCACCA CTGCCCTCCA GCCTGGGCAA CAGAGCAAGA 26580

CTCCGTCTCA AAAAAAAAAA AATGCTGCCC AAGCTGTGTT TGCACCACTG CCCTCCAGCC 26640 TGGGCAACAG AGCAAGACTC CGTCTCAAAA AAAAAAAAAA AAAATGCTGC CCAAGCTGTG 26700

TTTGCACCAC TGCCCTCCAG CCTGGGCAAC AGAGCAAGAC TCTGTCTCAA AAAAAAAAAA 26760

AATGCTGCCC AAGCTGTGTT TGCACCACTG CCCTCCGGCC TGGGCAACAG AGCAAGACTC 26820

CGTCTCAAAA AAAAAAAAAA AATGCTGCCC AAGCTGTGTT TGCACCACTG CCCTCCAGCC 26880

TGGGCAACAA AGCAAGCCTC AGCTTTCTGC CATCTCCACA ACCAAGAAAG CAATTCACAC 26940 AGAAATCAGT GCATCGTGCA GTGACCTCTT CAGAAAACCA ATGAGTTTTC CACCTGAGGA 27000

ACTGTTTCTG AGCCCCATTC AGAAAAACAC ATCCCTGTAA CTGCAGGGCA GATTTACTCA 27060

CTGTATGCCT GTTTAAATAA AGCTTCCAGC CTCTGCATGG GGTCTGTCTG GAAGCTCCTG 27120

TATCTGTCCC ACATTCTTGG AATCACAATG CACCCTTGGG AGGAAGATAT GTATTTAAAG 27180

GGAGTGGATG TTATGGTGAG AAAATGCTGC CCATCCTTCT AGAAGACAAA AGCCACACAA 27240 AATACATCAC AAGAACCAGT TTTTTTCAGA GAAGAACCTG CACAAAGAAC CTGCTCCCCC 27300

CACACCCCCA CACACAGGTG AATTAACAGG ATGTATGTTT TATCATAAAA GCACAGGTTT 27360 GTTTCCTATG CACTCTCTGA GGATTTGGCC ATATGCAAAG ATGTACAAAA ACCTTCTCTT 27420

TCCCCAGGGA ACCGTAACCC GTCTGAAAAG ATGCCCTTCT CAGAAGCGAG TTGAACGATT 27480

GTTGGAAAAG ATAAAATACG ACGTGCACAC ACACAGTAGA GAAATGTCAC CCATGCAAAT 27540

TATGTGTTTG AATGGAACAC ATTCAGGAAG CTAAATGGGG TATGACCACA CATTTGGGTT 27600 GATTTATTTG ACGAGTGGAA GGGGCAGATG GAAATGAATA CTGCTGTTTT CCTTTGGAAG 27660

GCCATATATG GGAATACCAA GAGGATTACT TTGGAAGTTT AGCTTCTCCA GGTGGTCTCT 27720

CTCTCTCTCT CTTTTTTTGA GACAGAGTCT CACTCTGTCA CCCAGGCTGC AGTGCAATGG 27780

CGTGCTCTCG GCTCACTGCA ACCTCAGCCT CCCAGGTACA AGCGATTCTC CTGCCTCAGC 27840

CTCCCGAGTA GCTGGGATCA CAGGTGTGCA CCACCACGCC TGGCTAATGT TTGTATTTTC 27900 AGTAGAGATG AGGTTTTACC ATGTTGGCCA GGCTGGTCTT GAACTCCTGA CCTCAGGTGA 27960

TCCGCCTGCC TCGGCCTCCC AAAGTGCTGG GATGACAGAC ATGAGCTAGC ACGCCCGGCC 28020

CCAGGTGGTC TTTTTAGCGG GTATTAAAGC AGCTTTCTCT CTGAGCCTTA AACCATGAAG 28080

ATAGACAGAC TCAGTGTATG GGTTTTAGAG TTGTAATTTT ATAAAAATAA GAAAAAGTCG 28140

ACCTATCATT GATGGTTAGT ATTTTTTGTA GCAGTTGCAT GCAATATTAG GATAAGGCAT 28200 GTTCTCAAAA AGAACTCTTT TTTTTTTTTT TTTGAGACGG AGTCTCGCTC TGTCACCCAG 28260

GCTGGAGTGC AGTGGCACGA TCTCCGCTCA CTGCAAGCTC CTCTTCCCGG GTTCACGCCA 28320

TTCTCCTGCC TCAGCCTCCC CAGTAGCTGG GACTACAGGC GCCCGCCACC ACGCCCGGCT 28380

AATTTTTTGT ATTTTTAGTA GAGACGGGGT TTCACCATGT TAGCCAGGAA GGTCTCGATC 28440

TCCTGACCTC ATGATCCGTC CGCCTCAGCC TCCCAAAGTG CTGGGACTAC AGGCGTGAGC 28500 CACTGCACTT GGCCTTTTTT TTTTTTTAGA TGGAGTTTTG CTCTTGTCGC CCAGGCTGGA 28560

GTATAATGGC ATGATCTCGA CTCACTGCAA CCTCCGCCTC CCGAGTTCAA GCGATTCTCC 28620

TGCCTCAGCC TCCCGAGTAG CTGGGATTAC AGGTGCCCAC CACCATGTCA AGATAATGTT 28680

TGTATTTTCA GTAGAGATGG GGTTTGACCA TGTTGGCCAG GCTGGTCTCG AACTCCTGAC 28740

CTCAGGTGAT CCACCCGCCT TAGCCTCCCA AAGTGCTGGG ATGACAGGCG TGAGCCCCTG 28800 CGCCCGGCCT TTGTAACTTT ATTTTTAATT TTTTTTTTTT TTTAAGAAAG ACAGAGTCTT 28860

GCTCTGTCAC CCAGGCTGGA GCACACTGGT GCGATCATAG CTCACTGCAG CCTCAAACTC 28920

CTGGGCTCAA GCAATCCTCC CACCTCAGCC TCCTGAGTAG CTGGGACTAC AGGCACCCAC 28980

CACCACACCC AGCTAATTTT TTTGATTTTT ACTAGAGACG GGATCTTGCT TTGCTGCTGA 29040

GGCTGGTCTT GAGCTCCTGA GCTCCAAAGA TCCTCTCACC TCCACCTCCC AAAGTGTTAG 29100 AATTACAAGC ATGAACCACT GCCCGTGGTC TCCAAAAAAA GGACTGTTAC GTGGATGTTC 29160

TAGCTTCCTG TTCTCGTCTT TTCTTTGTTA ATTGTACAGT TTGAGGGTGT GTGTGCGTGT 29220

GCGCACGTGT GTGTGTGCAG TCTCCTGATT TCATGTATTT AATTGTTATT ACCACCACCT 29280

CCATCTCTCA TTCCTTCTTA CCCTCACTGT GTAAAGATAC ATGTTGTTTT TAAATTTTAT 29340 GTATTTATAT TTATTTATTT GTATTTCTGA GACAGAGTCT CACTCTGTTG CCCAGGCTAG 29400

TGGCATGATC TCAGCTCACA GCAACCTTTG CCTCCTGGGT TCAAGCGATT CTCCTGCCTC 29460

AGCCTCCCGA GTAGCTGAGA TTACAGGCAC ACACCACCAC ACCCGGCTAG TTTTGTTTTG 29520

AGACGGAGTC TCGCTCTGTT GCAGGCTGCA GTGCAGTGGC GTGATCCTGG CTCACTGCAA 29580

CCTCTGCCTC CTGGATTCAA GCGATTCTCC TGCCTCAGCC TCCCAAGTAG CTGGGATTAC 29640

AGGCGCCCAC CGCCACACCT GGCTAATTTT TTATTGGTAG TAGAGACGGG GTTTCTCCAT 29700

GTTGACCAGA CTGGTCTTGA ACTCCCAACC TCGGGTGATC CACCCACCTG GGCCTCCCAA 29760 AGTGCTGGGA TGACAGGCGA GGGCCACCGC GTCCAGCCTT CTTCTTCTTC TTCTTTTTTT 29820

TTTTTTTAAG ATGGAGTTTC ACTCTGTTGC CCAGGCTGGA GTGCAGTGGT GCAATCTCGG 29880

CTCCCTGCAA CCTCCACCTC CCAGGTTCAA GAAATTCTTT TGCCTCAGCC TCCCGAGTAG 29940

CTGGGACTAC AGGTGCCCGC CACCACACCC ACCTAATGTT TGTATTTTTT TGGTAGAGAC 30000

GGGGCTTCAC CACATTGGCC AGGCTGGTCT TGAACTCCTG ACTTCAGATG ATCCTCCTGC 30060 CTCAGCCTCC CAGAGTGTTG GGATTACAGG CGTGAGCCAC GGTGCCCGGC CAGACGTCAT 30120

GTCTTAGGAA ATCAGAAAGT GGGTAGTTTC CGCACTCTGA GGAGAAAAAG AGACGTCCGG 30180

CGAAGAGAAA GGAGAGTGAA AGGATGTCTC CTCTTGTCTG TAGCCTGTTC TCAATCGTGA 30240

GTGAGCCAAT TGCCAGAAAC TGAGGGTGCT TCATTTGGCC AGGCAAGCTT CTCAACAGAA 30300

TGTCTAAGTA CTTGTTAATG CTGAGAAGCT CTCCAAGCTA CTGCACTCCA GCCTGGGTGA 30360 CAGAGCACGA CCTTGTCTGA AAACAATTAA TTAATCAATT AATTAATATA ATGAAATCAT 30420

ACTGAACTCA GGAGACCATT GGGGTGGGCA GGGCTGGGGT TGGAAAGGAA CATAAAATAT 30480

GGTGCAATGG ACTTTGCTCC AGTCTCCCTC CCCATCTCTT CTCGCCAAGA GTCTCTGGAG 30540

GGAGCATGGG GAAGATGCTT TGGGAATCTG TAACTTCTTG TCTTGTAAAC AGAATATCTA 30600

AGTAATTGTT AATGCTGAGA AGTTATAGAT TTCCAAAGCC TTTCTCCAGG CTACGGACAA 30660 GGGTCATGGG TTACTCAGTG TTACAGAAAG AATGACATGG AGATGTTTGT TACATCTTAA 30720

GGAACCATGA GGGGCCAGAG TATTTTACTC TAAGTGTAGA TGGTACATTG GCCACGCCTG 30780

TCCCAACACC ACCAATGGTG GCACCTAACT TTTGTGTTTG TGCCCCACAT TTCTTCTTCT 30840

TTTCTGACGT AAATGCAAGT GATATTCCTT GGAAACCATG CTGCAGCAAG AGGCCATCTG 30900

ACTACTAGTG ATACCCTGTA GCTCACCTAC AGCAGCTCAC TTGAAGCAGC TCACCCATAG 30960 CTCAGGTATA GCTCACCTGC AGCGGCTCAC CTGTAGCTCA CGTGTAGCTC ACTTGTAGCA 31020

GCTCACTGGT AGCTCACCTG CAGCAGCTCA CCTGTACCTC ACCTGTACCT CACCTGCAGC 31080

AGCTCACCTG TAGCTCACCT GTACGTGAGC CACCGTACCC GGCCAGCAAG ACCCCATTTC 31140

TAAAATAAAT ACACAAAAAT TAGCCGGACG CGGTGGCGCG TGTCTGTAGT TGTAGCTACT 31200

CAGGAGGCTG AGGTGGGAGG ATTGCTGGAG GCTGGGAGGT AGAGGCTGCA GTGAACCGTG 31260 ATCCAGCCAC TGTACTCTAG CCTGGATGAC ATAGCAAAAC CTTGTCTCAA AAAACAAAAA 31320

CAAAAAACAA AACAAAGAAA CAAACAAAAA ACCCACACAC ACCGGAAAAC AAAACAAAAA 31380 GCAAAAAGGA AAGAAAAGAG AGCCAGGTCC CAAATATATA TTTCCTTGGA GAACCATTTG 31440

CAAAGAGCAC ACTTAAGGCC GGGCGCGGTG GCTCACGCCT GTCATCCCGG CACTTTGGGA 31500

GGCCGAGGTG GGTGGATCAC GAGGTTGGGA GATCGAGACC ATCCTGGCCA ACATGGCGAA 31560

ACCCCATCTC TACTAAAAAT ACAAAAAATC AGCCAGGTGC TGAGGCAGGT GCCTGTAGTC 31620 CCAGCCACTC AGGAGGCTGA GGCAGGAGAA TGGCATGAAC CTGGGAGGTG GAGGTTGCAG 31680

TGAGCCGAGA TCGCGCCCCT GCACTCCAGC CTGGGCGACA GAGCGAGACT CCTTCTCAAA 31740

TAAATAAATA AATAAATAAC AAAGAGCAAA CTTAAAATTG TCTCAGAAAT CCCACGGGAT 31800

ATTGGATCTC CCTCATGCCT ATCTGATGAC ACTTTGAGTG TCTGGGGCCC CGTGCCTATT 31860

TTCTGGGGTT CCCAGAAGCT GCCGTTCTGA AAGTGTGGCT CTCGGGGACG TGGCACAGGT 31920 GTGGATGTCT GTTTTAAATG TCAGGCGTTT GGACGTTGAG GAACGTGAGG CTGAAGGTCG 31980

CCTTCGCCGA CCCCCTGAGT TTAGGGTCCT GCCTTTTAAA ATCTTCCCAG CACTCTGTTG 32040

TTCACGCAAG CGTCCCATCT GTTTGGGTGG CCGTGCCGTC TGCATCTGTC TCGAACCTTC 32100

ACAGCTTTGC AGAATATCCT GTTTCTCAAT ACGGATGGAG AAACACGAGA CGCGTTTTCT 32160

GGGTTATTTT AGCCGTCACG GAGAACCCCA GACTCATGTG TGCTAATGAC CTCATTAATG 32220 ATACTCTGAG GCAGACAGCC CTGCCTGATC TTAACAACAT TTTTTAAATT TCTTTTTTTG 32280

TTGTTGTTGT TACAGCATCA TTCATATAAC GTAGGAAACC GTGATCAGTA GCTTTTAGGA 32340

TATTTGCAAC AGGGTGTAAC ADAAABD 32367 (2) INFORMATION FOR SEQ ID NO: 15:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 806 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: smgle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "SHOT"

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 43..615

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GTGTCCCCGG AGCTGAAAGA TCGCAAAGAG GATGCGAAAG GGATGGAGGA CGAAGGCCAG 60

ACCAAAATCA AGCAGAGGCG AAGTCGGACC AATTTCACCC TGGAACAACT CAATGAGCTG 120

GAGAGGCTTT TTGACGAGAC CCACTATCCC GACGCCTTCA TGCGAGAGGA ACTGAGCCAG 180

CGACTGGGCC TGTCGGAGGC CCGAGTGCAG GTTTGGTTTC AAAATCGAAG AGCTAAATGT 240

AGAAAACAAG AAAATCAACT CCATAAAGGT GTTCTCATAG GGGCCGCCAG CCAGTTTGAA 300 GCTTGTAGAG TCGCACCTTA TGTCAACGTA GGTGCTTTAA GGATGCCATT TCAGCAGGTT 360

CAGGCGCAGC TGCAGCTGGA CAGCGCTGTG GCGCACGCGC ACCACCACCT GCATCCGCAC 420 CTGGCCGCGC ACGCGCCCTA CATGATGTTC CCAGCACCGC CCTTCGGACT GCCGCTCGCC 480

ACGCTGGCCG CGGATTCGGC TTCCGCCGCC TCGGTAGTGG CGGCCGCAGC AGCCGCCAAG 540

ACCACCAGCA AGGACTCCAG CATCGCCGAT CTCAGACTGA AAGCCAAAAA GCACGCCGCA 600

GCCCTGGGTC TGTGACVCCA ACGCCAGCAC CAATGTCGCG CCTGTCCCGC GGCACTCAGC 660 CTGCASNCCC TNDDKANMCG TTRCTYHTCM ATTACACTTT GGGACCYCGG GDBAGVCCTT 720

TTNNAGACTT YVATKGGSC CSCTGGBCCC TBRKGAWAC TTGSGHYCGR GAACCGAKHT 780

GCCCABAYGA GGACCRGTTT GGAKDG 806

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 190 ammo acids (B) TYPE: ammo acid

(C) STRANDEDNESS: smgle

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Met Glu Asp Glu Gly Gin Thr Lys lie Lys Gin Arg Arg Ser Arg Thr 1 5 10 15

Asn Phe Thr Leu Glu Gin Leu Asn Glu Leu Glu Arg Leu Phe Asp Glu 20 25 30

Thr His Tyr Pro Asp Ala Phe Met Arg Glu Glu Leu Ser Gin Arg Leu 35 40 45 Gly Leu Ser Glu Ala Arg Val Gin Val Trp Phe Gin Asn Arg Arg Ala 50 55 60

Lys Cys Arg Lys Gin Glu Asn Gin Leu His Lys Gly Val Leu lie Gly 65 70 75 80

Ala Ala Ser Gin Phe Glu Ala Cys Arg Val Ala Pro Tyr Val Asn Val 85 90 95

Gly Ala Leu Arg Met Pro Phe Gin Gin Val Gin Ala Gin Leu Gin Leu 100 105 110

Asp Ser Ala Val Ala His Ala His His His Leu His Pro His Leu Ala 115 120 125 Ala His Ala Pro Tyr Met Met Phe Pro Ala Pro Pro Phe Gly Leu Pro 130 135 140

Leu Ala Thr Leu Ala Ala Asp Ser Ala Ser Ala Ala Ser Val Val Ala

145 150 155 160

Ala Ala Ala Ala Ala Lys Thr Thr Ser Lys Asp Ser Ser lie Ala Asp

165 170 175

Leu Arg Leu Lys Ala Lys Lys His Ala Ala Ala Leu Gly Leu 180 185 190

Claims

Claims

An isolated human nucleic acid molecule encoding polypeptides containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID

NO 1 and having regulating activity on human growth

A nucleic acid molecule according to claim 1 which is selected from the following group a) an isolated DNA molecule comprising a nucleotide sequence (i) encoding a polypeptide containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID NO 1 and which has the biological activity to regulate human growth, or (ii) encoding a polypeptide containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID NO 1 except that one or more amino acid residues have been deleted, added or substituted but which retains the same biological activity of regulating human growth, b) an isolated DNA molecule comprising the nucleotide sequence of SHOX ET93 [SEQ ID NO 2] and the nucleotide sequence of SHOX ET45 [SEQ ID NO 4] or fragments thereof, c) nucleic acid molecules capable of hybridizing to the DNA molecules of a) or b), and d) DNA molecules comprising a nucleotide sequence having a homology of seventy percent or higher with the DNA molecules of a) or b)

A DNA molecule according to claim 2 which encodes a polypeptide having an N- terminal and/or C-terminal amino acid extension to the homeobox domain of sixty amino acids of SEQ ID NO 1

A DNA molecule according to claim 3 which encodes a polypeptide having a length of 150 to 350 amino acids

A DNA molecule according to any of claims 2 - 4 further comprising the nucleotide sequence of SHOX G310 [SEQ ID NO 3]

A DNA molecule according to any of claims 2 - 5 further comprising the nucleotide sequence of SHOX G108 [SEQ ID NO 5] 7 A DNA molecule according to any of claims 2 - 6 further comprising the nucleotide sequence of SHOX Va [SEQ ID NO 6] or SHOX Vb [SEQ ID NO 7]

8 A DNA molecule according to any of claims 1 - 4 which encodes a polypeptide which is selected from the following group a) transcription factor A having essentially the amino acid sequence of [SEQ ID NO 11], b) transcription factor B having essentially the amino acid sequence of [SEQ ID NO 13], and c) transcription factor C having essentially the amino acid sequence of [SEQ ID NO 15]

9 DNA sequence comprising the nucleotide sequence of SHOX ET93 [SEQ ID No 2]

10 A DNA sequence according to claim 9 further comprising the nucleotide sequence of SHOX G310 [SEQ ID NO 3]

11 A DNA sequence according to claim 9 or 10 further comprising the nucleotide sequence of SHOX ET45 [SEQ ID NO 4]

12 A DNA sequence according to any of claims 9 - 12 further comprising the nucleotide sequence of SHOX G108 [SEQ ID 5]

13 A DNA sequence according to any of claims 9 - 12 further comprising either the nucleotide sequence of SHOX Va [SEQ ID 6] or SHOX Vb [SEQ ID 7]

14 A DNA sequence according to claim 9 comprising the nucleotide sequence of SHOX ET93 [SEQ ID No 2] and the nucleotide sequence of SHOX ET45 [SEQ

ID No 4]

15 A DNA sequence according to claim 9 comprising the nucleotide sequence of SHOX ET93 [SEQ ID NO 2], the nucleotide sequence of SHOX ET45 [SEQ ID No 4] and the nucleotide sequence of SHOX G108 [SEQ ID 5] 16 A DNA sequence according to any of claims 9 - 15 comprising the nucleotide sequences of SHOX G310 [SEQ ID NO 3], SHOX ET93 [SEQ ID NO 2], SHOX ET45 [SEQ ID No 4] and SHOX G108 [SEQ ID 5]

17 A DNA sequence according to claim 17 further comprising the nucleotide sequence of SHOX Va [SEQ ID No 6]

18 A DNA sequence according to claim 16 further comprising the nucleotide sequence of SHOX Vb [SEQ ID No 7]

19 A DNA sequence according to claim 9 consisting essentially of the isolated genomic sequence of the PAR1 region identified in [SEQ ID No 14]

20 A DNA sequence comprising the nucleotde sequence of SHOX ET92 [SEQ ID No 9]

21 A DNA sequence according to any of claims 9 - 20 whereby the DNA is a genomic or isolated DNA responsible for regulating human growth

22 A DNA sequence according to any of claims 9 - 21 whereby the DNA is a cDNA

23 A cDNA according to claim 22 consisting essentially of the nucleotide sequence of SHOXa [SEQ ID No 10] or SHOXb [SEQ ID NO 12]

24 A cDNA according to claim 22 consisting essentially of the nucleotide sequence of SHOT [SEQ ID No 14]

25 A human growth protein (transcription factor SHOXa) having the amino acid sequence given in [SEQ ID No 11] or a functional fragment thereof

26 A human growth protein (transcription factor SHOXb) having the amino acid sequence given in [SEQ ID No 13] or a functional fragment thereof

27 A human growth protein (transcription factor SHOT) having the amino acid sequence give in [SEQ ID NO 15] or a functional fragment thereof

28 A cDNA encoding for a protein according to claim 25, 26 or 27 29 A pharmaceutical composition comprising a protein according to any of claims 25 to 27

30 A method for the treatment of short stature comprising administering to a subject in need thereof a therapeutically effictive amount of a protein according to claim 25 to 27

31 Use of a protein according to claim 25 to 27 for the preparation of a pharmaceutical composition for the treatment of short stature

32 Use of a DNA sequence according to claims 1 - 24 for the preparation of a pharmaceutical composition for the treatment of disorders relating to mutations of the short stature gene

33 Use of a DNA sequence according to any of claims 1 - 24 for the preparation of a kit for the identification of individuals having a genetic defect responsible for deminished human growth

34 Use of a DNA sequence according to claim 33 for the identification of a gene responsible for short human stature

35 Method for the determination of short stature on the basis of RNA or DNA molecules, wherein the biological sample molecule to be examined is amplified in the presence of two nucleotide probes completely or in part complementary to any of the DNA sequences mentioned in SEQ ID No 2 to SEQ ID No 7 and subsequently determined by a suitable detection system

36 Use of the method according to claim 35 for the identification of persons having a genetic defect responsible for short stature

37 Transgenic animal transformed with a gene responsible for short stature containing a DNA sequence according to any one of claims 1 - 24

38 Cells transformed with a DNA sequence according to any one of claims 1 - 24

39 Test system for identifying or screening pharmaceutical agents useful for the treatment of human short stature comprising a cell according to claim 38 Method for identifying or screening of candidates for pharmaceutical agents useful for the treatment of disorders relating to mutations in the short stature gene comprising providing a test system according to claim 39 and determining variations in the phenotype of said cells or variations in the expression products of said cells after contacting said cells with said candidate pharmaceutical agents

An expression vector comprising a DNA molecule according to claims 1 - 8 which is capable of effecting the expression of the encoded polypeptide

A method for the in vivo treatment of human growth disorders related to at least one mutation in the SHOX or SHOT gene by gene therapy, compπsing introducing mto human cells an expression plasmid in which a DNA molecule according to any of claims 1 - 8 is incorporated downstream from the expression promotor that effects expression in a human host cell

A method according to claim 42 for the treatment of Turner syndrome or short stature

Antibodies obtained by immunization of mammals using the transcription factors A, B or C or antigenic fragments thereof and isolating such antibodies from such mammals